A , D The indicated clones and the parental cell line ( WT ) were transiently transfected with the RARE - DR5 - Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle ( DMSO ) and ATRA ( 100 nM ) , as illustrated in the upper bar graph . Each value is the mean ± SD of three replicate cultures . The levels of the RARαprotein measured in the indicated clones by Western blot analysis is shown under the bar graph . To demonstrate that similar levels of total proteins were loaded in each lane , the β - actin band signal obtained after re - blotting of the gel is shown . FI = Fluorescence intensity . B , E The panels illustrate the growth curves of the indicated MDA - MB453 and SKBR3 clones and the SKBR3 parental cell lines ( WT ) measured with the sulforhodamine assay . C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA - MB453 clones and the parental cell line . The cell lines were challenged with vehicle ( DMSO ) or ATRA for 3 , 6 , and 9 days prior to the sulforhodamine assay . OD = optical density at 540 nm . Each value is the mean ± SD of five replicate culture wells .

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

A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARαprotein measured in the indicated clones by Western blot analysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.


A , D The indicated clones and the parental cell line ( WT ) were transiently transfected with the RARE - DR5 - Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle ( DMSO ) and ATRA ( 100 nM ) , as illustrated in the upper bar graph . Each value is the mean ± SD of three replicate cultures . The levels of the RARαprotein measured in the indicated clones by Western blotanalysis is shown under the bar graph . To demonstrate that similar levels of total proteins were loaded in each lane , the β - actin band signal obtained after re - blotting of the gel is shown . FI = Fluorescence intensity . B , E The panels illustrate the growth curves of the indicated MDA - MB453 and SKBR3 clones and the SKBR3 parental cell lines ( WT ) measured with the sulforhodamine assay . C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA - MB453 clones and the parental cell line . The cell lines were challenged with vehicle ( DMSO ) or ATRA for 3 , 6 , and 9 days prior to the sulforhodamine assay . OD = optical density at 540 nm . Each value is the mean ± SD of five replicate culture wells .

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

A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARαprotein measured in the indicated clones by Western blotanalysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.


F The graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated SKBR3 clones and the parental cell line . The cell lines were challenged with vehicle ( DMSO ) or ATRA for 3 and 6 days prior to the sulforhodamine assay . Each value is the mean ± SD of five replicate culture wells .

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 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

F The graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated SKBR3 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3 and 6 days prior to the sulforhodamine assay. Each value is the mean ± SD of five replicate culture wells.


G The panel illustrates the effects exerted by ATRA ( 0 . 1 μM ) on random cell motility of the indicated MDA - MB453 and SKBR3 clones . The results are representative of two independent experiments . Each value is the mean ± SE of the motility of at least 60 cells .

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

G The panel illustrates the effects exerted by ATRA (0.1 μM) on random cell motility of the indicated MDA-MB453 and SKBR3 clones. The results are representative of two independent experiments. Each value is the mean ± SE of the motility of at least 60 cells.


B , C GOT2 can be acetylated . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T cells treated with NAM ( 5 mM ) and / or TSA ( 0 . 5 mM ) for the indicated time period . Acetylation levels of Flag ‐ bead ‐ purified GOT2 were determined by Western blot analysis using a pan ‐ anti ‐ acetyllysine antibody ( α ‐ Ac ) . IB and IP denote immunoblotting and immunoprecipitation , respectively . Relative GOT2 acetylation ratios were calculated after normalizing against Flag .

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 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 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

B, C GOT2 can be acetylated. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period. Acetylation levels of Flag‐bead‐purified GOT2 were determined by Western blot analysis using a pan‐anti‐acetyllysine antibody (α‐Ac). IB and IP denote immunoblotting and immunoprecipitation, respectively. Relative GOT2 acetylation ratios were calculated after normalizing against Flag.


D GOT2 acetylation enhances the interaction between ectopically expressed GOT2 and MDH2 . Flag ‐ GOT2 and Myc ‐ MDH2 were co ‐ overexpressed in HEK293T cells treated without or with NAM ( 5 mM ) for the indicated time period . The acetylation level of Flag ‐ bead ‐ purified GOT2 and the protein association between ectopic proteins of GOT2 and MDH2 were determined by Western blot analysis .

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D GOT2 acetylation enhances the interaction between ectopically expressed GOT2 and MDH2. Flag‐GOT2 and Myc‐MDH2 were co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐bead‐purified GOT2 and the protein association between ectopic proteins of GOT2 and MDH2 were determined by Western blot analysis.


E Acetylation enhances the interaction between ectopically expressed GOT2 and endogenous MDH2 . Flag ‐ GOT2 was co ‐ overexpressed in HEK293T cells treated without or with NAM ( 5 mM ) for the indicated time period . The acetylation level of Flag ‐ GOT2 and its association with endogenous MDH2 were determined by Western blot analysis .

O O O 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

E Acetylation enhances the interaction between ectopically expressed GOT2 and endogenous MDH2. Flag‐GOT2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐GOT2 and its association with endogenous MDH2 were determined by Western blot analysis.


F Acetylation enhances the interaction between ectopically expressed MDH2 and endogenous GOT2 . Flag ‐ MDH2 was co ‐ overexpressed in HEK293T cells treated without or with NAM ( 5 mM ) for the indicated time period . The protein association between Flag ‐ MDH2 and endogenous GOT2 was determined by Western blot analysis .

O O O 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

F Acetylation enhances the interaction between ectopically expressed MDH2 and endogenous GOT2. Flag‐MDH2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The protein association between Flag‐MDH2 and endogenous GOT2 was determined by Western blot analysis.


H Mapping the major lysine residue ( s ) of acetylation in GOT2 whose acetylation can affect protein interaction between GOT2 and MDH2 . Putative acetylated residues were divided into six groups according to their position in the structure of GOT2 . Each group of putative acetylated lysine ( K ) sites was mutated to arginine ( R ) , and the deacetylated mimic K ‐ to ‐ R mutants were examined for their protein association with ectopically expressed Myc ‐ MDH2 by Western blot analysis .

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 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

H Mapping the major lysine residue(s) of acetylation in GOT2 whose acetylation can affect protein interaction between GOT2 and MDH2. Putative acetylated residues were divided into six groups according to their position in the structure of GOT2. Each group of putative acetylated lysine (K) sites was mutated to arginine (R), and the deacetylated mimic K‐to‐R mutants were examined for their protein association with ectopically expressed Myc‐MDH2 by Western blot analysis.


I GOT2 coupled with MDH2 shows a high level of K159 acetylation . Flag ‐ GOT2 was overexpressed in HEK293T cells without or with co ‐ overexpression of Myc ‐ MDH2 . These transfected cells were treated with NAM ( 5 mM ) for 5 h . Double immunoprecipitation was performed to obtain the Flag ‐ GOT2 from the Myc ‐ MDH2 immunoprecipitates ( Ppt , supposed to be GOT2 bound with MDH2 ) . The remaining supernatants ( Sup , supposed to be GOT2 not bound with MDH2 ) were also harvested and precipitated by Flag antibody to test the K159 acetylation level of Flag ‐ GOT2 by Western blot analysis .

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

I GOT2 coupled with MDH2 shows a high level of K159 acetylation. Flag‐GOT2 was overexpressed in HEK293T cells without or with co‐overexpression of Myc‐MDH2. These transfected cells were treated with NAM (5 mM) for 5 h. Double immunoprecipitation was performed to obtain the Flag‐GOT2 from the Myc‐MDH2 immunoprecipitates (Ppt, supposed to be GOT2 bound with MDH2). The remaining supernatants (Sup, supposed to be GOT2 not bound with MDH2) were also harvested and precipitated by Flag antibody to test the K159 acetylation level of Flag‐GOT2 by Western blot analysis.


J The 3KQ / R mutations do not change GOT2 enzyme activity . Wild ‐ type and 3K mutant GOT2 proteins were overexpressed and purified from E . coli , and the enzyme activity of GOT2 was determined as described in " " . Shown are average values with standard deviation ( SD ) of triplicated experiments . n . s . = not significant for the indicated comparison .

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

J The 3KQ/R mutations do not change GOT2 enzyme activity. Wild‐type and 3K mutant GOT2 proteins were overexpressed and purified from E. coli, and the enzyme activity of GOT2 was determined as described in "". Shown are average values with standard deviation (SD) of triplicated experiments. n.s. = not significant for the indicated comparison.


A , B Glucose and glutamine increase GOT2 K159 acetylation and GOT2 - MDH2 association in HEK293T cells . Flag ‐ GOT2 and Myc ‐ MDH2 were overexpressed in cells treated with increased concentrations of glucose ( A ) or glutamine ( B ) for 6 h . GOT2 proteins were purified by Flag beads , and the K159 acetylation level of GOT2 and its protein association with Myc ‐ MDH2 were determined by Western blot analysis .

O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

A, B Glucose and glutamine increase GOT2 K159 acetylation and GOT2-MDH2 association in HEK293T cells. Flag‐GOT2 and Myc‐MDH2 were overexpressed in cells treated with increased concentrations of glucose (A) or glutamine (B) for 6 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with Myc‐MDH2 were determined by Western blot analysis.


C , D Glucose and glutamine increase GOT2 K159 acetylation in Panc ‐ 1 cells . Flag ‐ GOT2 was overexpressed in cells treated with different concentrations of glucose ( C ) and glutamine ( D ) for 4 h . GOT2proteins were purified by Flag beads , and the K159 acetylation level of GOT2 was determined by Western blot analysis . Relative GOT2 K159 acetylation levels were normalized against Flagprotein levels .

O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

C, D Glucose and glutamine increase GOT2 K159 acetylation in Panc‐1 cells. Flag‐GOT2 was overexpressed in cells treated with different concentrations of glucose (C) and glutamine (D) for 4 h. GOT2proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis. Relative GOT2 K159 acetylation levels were normalized against Flagprotein levels.


E , F Quantification of the percentage of K159 ‐ acetylated endogenous GOT2 in Panc ‐ 1 cells . Recombinant fully K159 ‐ acetylated GOT2 was loaded onto the same gel , together with endogenous GOT2 from Panc ‐ 1 cells treated without or with glucose ( 12 mM ) ( E ) or glutamine ( 2 mM ) ( F ) for 4 h . GOT2 protein and K159 acetylation were detected by Western blot . The percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 protein levels .

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

E, F Quantification of the percentage of K159‐acetylated endogenous GOT2 in Panc‐1 cells. Recombinant fully K159‐acetylated GOT2 was loaded onto the same gel, together with endogenous GOT2 from Panc‐1 cells treated without or with glucose (12 mM) (E) or glutamine (2 mM) (F) for 4 h. GOT2 protein and K159 acetylation were detected by Western blot. The percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 protein levels.


G , H 3K mutant GOT2 displays negligible response in changing protein association with MDH2 after glucose or glutamine treatment . Panc ‐ 1 cells with GOT2 knockdown and re ‐ expression of wild ‐ type or 3K mutant GOT2 were treated with glucose ( G ) or glutamine ( H ) at the indicated concentrations for 4 h . The protein association between Flag ‐ tagged wild ‐ type or 3K mutant GOT2 and endogenous MDH2 was determined by Western blot analysis .

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

G, H 3K mutant GOT2 displays negligible response in changing protein association with MDH2 after glucose or glutamine treatment. Panc‐1 cells with GOT2 knockdown and re‐expression of wild‐type or 3K mutant GOT2 were treated with glucose (G) or glutamine (H) at the indicated concentrations for 4 h. The protein association between Flag‐tagged wild‐type or 3K mutant GOT2 and endogenous MDH2 was determined by Western blot analysis.


A GOT2 interacts with SIRT3 , but not SIRT4 and SIRT5 . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T cells together with the individual HA ‐ tagged SIRT as indicated . Proteins were purified by IP with Flag beads , following Western blot to detect SIRTs with an HA antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 GOT2 interacts with SIRT3, but not SIRT4 and SIRT5. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells together with the individual HA‐tagged SIRT as indicated. Proteins were purified by IP with Flag beads, following Western blot to detect SIRTs with an HA antibody.


B Endogenous proteins of GOT2 and SIRT3 interact with each other . SIRT3 protein in HEK293T cells was purified by IP with an anti ‐ SIRT3 antibody , following Western blot to detect GOT2 with an anti ‐ GOT2 antibody .

O O O O O O O O O O O O O O O O O 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 Endogenous proteins of GOT2 and SIRT3 interact with each other. SIRT3 protein in HEK293T cells was purified by IP with an anti‐SIRT3 antibody, following Western blot to detect GOT2 with an anti‐GOT2 antibody.


C GOT2 deacetylation is dependent on SIRT3 catalytic activity . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T cells together with HA ‐ tagged wild ‐ type SIRT3 and a catalytically inactive mutant , SIRT3 H248Y . GOT2 proteins were purified by Flag beads , following Western blot to detect GOT2 K159 acetylation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 GOT2 deacetylation is dependent on SIRT3 catalytic activity. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells together with HA‐tagged wild‐type SIRT3 and a catalytically inactive mutant, SIRT3 H248Y. GOT2 proteins were purified by Flag beads, following Western blot to detect GOT2 K159 acetylation.


D SIRT3 impairs the protein interaction between GOT2 and MDH2 . HEK293T cells were transfected with the plasmids as indicated . GOT2 proteins were purified by Flag beads , and their protein interaction with Myc ‐ MDH2 was determined by Western blot .

O O O O O O O O O O O O O O O O O O O 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 SIRT3 impairs the protein interaction between GOT2 and MDH2. HEK293T cells were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and their protein interaction with Myc‐MDH2 was determined by Western blot.


E Knockdown of SIRT3 increases GOT2 K159 acetylation and enhances GOT2 - MDH2 association . HEK293T cells with or without transient SIRT3 knockdown were transfected with the plasmids as indicated . GOT2 proteins were purified by Flag beads , and the K159 acetylation level of GOT2 and its protein association with endogenous MDH2 were determined by Western blot analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Knockdown of SIRT3 increases GOT2 K159 acetylation and enhances GOT2-MDH2 association. HEK293T cells with or without transient SIRT3 knockdown were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with endogenous MDH2 were determined by Western blot analysis.


F , G Knockdown of SIRT3 diminishes the effect of glucose or glutamine on changing GOT2 K159 acetylation . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T cells without or with transient SIRT3 knockdown . These cells were treated with different concentrations of glucose ( F ) and glutamine ( G ) as indicated . GOT2 proteins were purified by Flag beads , and the K159 acetylation level of GOT2 was determined by Western blot analysis .

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

F, G Knockdown of SIRT3 diminishes the effect of glucose or glutamine on changing GOT2 K159 acetylation. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells without or with transient SIRT3 knockdown. These cells were treated with different concentrations of glucose (F) and glutamine (G) as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis.


H , I Up ‐ regulation of Sirt3 leads to decreased Got2 K159 acetylation in mouse tissues during fasting . C57BL / 6 mice ( male , 20 weeks ; n = 2 per group ) were fed a normal chow diet or fasted overnight . Upon sacrifice , mouse liver and white adipose tissues were harvested , and the levels of the indicated proteins were determined by Western blot analysis .

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

H, I Up‐regulation of Sirt3 leads to decreased Got2 K159 acetylation in mouse tissues during fasting. C57BL/6 mice (male, 20 weeks; n = 2 per group) were fed a normal chow diet or fasted overnight. Upon sacrifice, mouse liver and white adipose tissues were harvested, and the levels of the indicated proteins were determined by Western blot analysis.


A The malate - aspartate shuttle , such as LDH , also plays a key role in regulating cytosolic NADH levels in Panc ‐ 1 cells . Cells were treated with AOA ( 1 . 6 mM , 20 min ) , oxamate ( 5 mM , 20 min ) , and EGCG ( 0 . 5 mM , 20 min ) . The cytosolic NADH level was measured as described in .

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A The malate-aspartate shuttle, such as LDH, also plays a key role in regulating cytosolic NADH levels in Panc‐1 cells. Cells were treated with AOA (1.6 mM, 20 min), oxamate (5 mM, 20 min), and EGCG (0.5 mM, 20 min). The cytosolic NADH level was measured as described in .


B , C GOT2 3K acetylation promotes the net transfer of cytosolic NADH into mitochondria . In Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins , the NADH level in the cytosol ( B ) and mitochondrion ( C ) was determined in cell extracts as described in .

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B, C GOT2 3K acetylation promotes the net transfer of cytosolic NADH into mitochondria. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the NADH level in the cytosol (B) and mitochondrion (C) was determined in cell extracts as described in .


D GOT2 3K acetylation regulates mitochondrial NADH / NAD + redox in the cell . In Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins , the ratio of NADH / NAD + in the mitochondria of cells was measured as described in .

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

D GOT2 3K acetylation regulates mitochondrial NADH/NAD+ redox in the cell. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADH/NAD+ in the mitochondria of cells was measured as described in .


E GOT2 3K acetylation promotes ATP production . In Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins , ATP production was determined in cell extracts as described in .

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 O O O O O O

E GOT2 3K acetylation promotes ATP production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ATP production was determined in cell extracts as described in .


F GOT2 3K acetylation promotes NADPH production . In Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins , the ratio of NADPH / NADP + was determined in cell extracts as described in .

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F GOT2 3K acetylation promotes NADPH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADPH/NADP+ was determined in cell extracts as described in .


G GOT2 3K acetylation promotes GSH production . In Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins , the GSH / GSSG ratio was determined in cell extracts as described in .

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

G GOT2 3K acetylation promotes GSH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the GSH/GSSG ratio was determined in cell extracts as described in .


H GOT2 3K acetylation suppresses cellular ROS levels . In Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins , ROS was determined as described in in cells under non ‐ stressed condition or exposed to menadione ( 50 μM for 30 min ) .

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H GOT2 3K acetylation suppresses cellular ROS levels. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ROS was determined as described in in cells under non‐stressed condition or exposed to menadione (50 μM for 30 min).


I GOT2 3K acetylation protects cells from oxidative damage . Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins were treated with increasing concentrations of menadione for 3 h as indicated , and cell viability was determined by counting the remaining adherent cells .

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I GOT2 3K acetylation protects cells from oxidative damage. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were treated with increasing concentrations of menadione for 3 h as indicated, and cell viability was determined by counting the remaining adherent cells.


J , K Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins were seeded in a 6 ‐ well plate , and the cells were maintained under the condition of low glucose ( 0 . 5 mM , J ) or high glucose ( 12 mM , K ) . Culture medium was refreshed every day , and cell numbers were counted every 1 - 2 days over a period of 7 days .

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J, K Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were seeded in a 6‐well plate, and the cells were maintained under the condition of low glucose (0.5 mM, J) or high glucose (12 mM, K). Culture medium was refreshed every day, and cell numbers were counted every 1-2 days over a period of 7 days.


L , M Panc ‐ 1 stable cells in ( J , K ) were grown under low glucose ( 0 . 5 mM ) or high glucose ( 12 mM ) for 7 days . The level of histone H3 phosphorylation at Ser10 was determined by Western blot analysis ( L ) . The percentages of living cells and apoptotic cells were determined by flow cytometry as described in ( M ) .

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L, M Panc‐1 stable cells in (J, K) were grown under low glucose (0.5 mM) or high glucose (12 mM) for 7 days. The level of histone H3 phosphorylation at Ser10 was determined by Western blot analysis (L). The percentages of living cells and apoptotic cells were determined by flow cytometry as described in (M).


A - C GOT2 3K acetylation promotes xenograft tumorgrowth . Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins ( 2 × 106cells ) were injected subcutaneously into the flanks of nude mice . The tumor volume was carefully monitored over the indicated time period ( A ) . At 8 weeks after injection , tumors from six mice were extracted , photographed , and weighted ( B , C ) .

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A-C GOT2 3K acetylation promotes xenograft tumorgrowth. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins (2 × 106cells) were injected subcutaneously into the flanks of nude mice. The tumor volume was carefully monitored over the indicated time period (A). At 8 weeks after injection, tumors from six mice were extracted, photographed, and weighted (B, C).


D GOT2 3K acetylation promotes tumor cellproliferation . Tumor sections from xenografts were prepared for IHC staining , and the PCNA ‐ stained tumor sections were analyzed by quantifying the PCNA ‐ positive area .

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D GOT2 3K acetylation promotes tumor cellproliferation. Tumor sections from xenografts were prepared for IHC staining, and the PCNA‐stained tumor sections were analyzed by quantifying the PCNA‐positive area.


E Clinical cases with increased GOT2 K159 acetylation in humanpancreatictumor tissues . 10 pairs of humanpancreatictumor tissue ( shown as T ) and adjacent normal tissue ( shown as N ) were lysed . The acetylation level of GOT2 at K159 was compared against GOT2protein by Western blot . F Clinical cases with decreased SIRT3protein expression in humanpancreatictumor tissues . 10 pairs of humanpancreatictumor tissue ( shown as T ) and adjacent normal tissue ( shown as N ) were lysed . The SIRT3protein levels were compared against tubulin by Western blot . G , H Quantification of GOT2 K159 acetylation and SIRT3protein expression in clinical cases as described in ( E ) and ( F ) .

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E Clinical cases with increased GOT2 K159 acetylation in humanpancreatictumor tissues. 10 pairs of humanpancreatictumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The acetylation level of GOT2 at K159 was compared against GOT2protein by Western blot.F Clinical cases with decreased SIRT3protein expression in humanpancreatictumor tissues. 10 pairs of humanpancreatictumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The SIRT3protein levels were compared against tubulin by Western blot.G, H Quantification of GOT2 K159 acetylation and SIRT3protein expression in clinical cases as described in (E) and (F).


B . DIS3 expression in total cell lysates of control ( siCTRL ) and DIS3 U2OS silenced cells ( siDIS3 ) was analyzed by Western blot upon rescue with empty vector ( EV ) or DIS3 expressing vector ( DIS3 ) . Lamin B was used as the loading control .

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B. DIS3 expression in total cell lysates of control (siCTRL) and DIS3 U2OS silenced cells (siDIS3) was analyzed by Western blot upon rescue with empty vector (EV) or DIS3 expressing vector (DIS3). Lamin B was used as the loading control.


C . Representative images of comet assays performed under alkaline conditions in U2OS cells transfected with siCTRL or siDIS3 and then infected with a vector expressing DIS3 ( DIS3 ) or an empty vector ( EV ) . Quantitative analysis of comet tail lengths for each condition . Plots represent three biological replicates , with more than 150 cells for each point . The analysis is performed using Casplab software , error bars indicate s . e . m . ( n = 3 ) . * * * * p - value < 0 . 0001 . ns = not statistically significant , two - way ANOVA , scale bar is 200 µm .

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C. Representative images of comet assays performed under alkaline conditions in U2OS cells transfected with siCTRL or siDIS3 and then infected with a vector expressing DIS3 (DIS3) or an empty vector (EV). Quantitative analysis of comet tail lengths for each condition. Plots represent three biological replicates, with more than 150 cells for each point. The analysis is performed using Casplab software, error bars indicate s.e.m. (n=3). **** p-value < 0.0001. ns= not statistically significant, two-way ANOVA, scale bar is 200 µm.


D . Immunostaining against RPA in U2OS cells transfected with siCTRL or with siDIS3 and 48 hours later infected with a vector expressing DIS3 ( DIS3 ) or an empty vector ( EV ) . The averages of foci number per cell were calculated from three independent experiments using Cell Profiler software . At least 150 cells were counted for each point , error bars indicate s . e . m . ( n = 3 ) . * p - value < 0 . 05 . ns = not statistically significant , two - way ANOVA , scale bar is 10 µm .

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D. Immunostaining against RPA in U2OS cells transfected with siCTRL or with siDIS3 and 48 hours later infected with a vector expressing DIS3 (DIS3) or an empty vector (EV). The averages of foci number per cell were calculated from three independent experiments using Cell Profiler software. At least 150 cells were counted for each point, error bars indicate s.e.m. (n=3). * p-value < 0.05. ns= not statistically significant, two-way ANOVA, scale bar is 10 µm.


A . Immunostaining with S9 . 6 ( green ) and DAPI ( blue ) in U2OS cells , transfected for 48 hours with siCTRL or siDIS3 and then mock - treated ( UNTR ) or treated with RNase H or RNase III for 1 hour before fixation . Histograms show S9 . 6 signal intensity per cell , error bars indicate s . e . m . ( n = 3 , biological replicates ) . More than 150 cells were counted , and error bars indicate s . e . m . ( n = 3 biological replicates ) . ) . * * * * = p < 0 . 0001 , * * * = p < 0 . 001 , ns = not statistically significant , two - way ANOVA , scale bar is 10 µm .

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A. Immunostaining with S9.6 (green) and DAPI (blue) in U2OS cells, transfected for 48 hours with siCTRL or siDIS3 and then mock-treated (UNTR) or treated with RNase H or RNase III for 1 hour before fixation. Histograms show S9.6 signal intensity per cell, error bars indicate s.e.m. (n=3, biological replicates). More than 150 cells were counted, and error bars indicate s.e.m. (n=3 biological replicates).). **** = p < 0.0001, *** = p < 0.001, ns = not statistically significant, two-way ANOVA, scale bar is 10 µm.


B . Immunostaining with J2 ( green ) and DAPI ( blue ) in U2OS cells , as in panel A ) . More than 150 cells were counted , and error bars indicate s . e . m . ( n = 3 biological replicates ) . For all conditions , differences between siCTRL and siDIS3 were not statistically significant , two - way ANOVA , scale bar is 10 µm .

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B. Immunostaining with J2 (green) and DAPI (blue) in U2OS cells, as in panel A). More than 150 cells were counted, and error bars indicate s.e.m. (n=3 biological replicates). For all conditions, differences between siCTRL and siDIS3 were not statistically significant, two-way ANOVA, scale bar is 10 µm.


C . Dot blot analysis of DNA : RNA hybrids formation : serial dilutions ( 750 ng , 375 ng , 187 , 5 ng , 93 , 75 ng , 46 , 87 ng ) of genomic DNA extracted from WT or DIS3 silenced U2OS cells and pre - treated with RNase H , RNase III or mock treated , were arrayed on a membrane and probed using the S9 . 6 antibody . The histogram shows the S9 . 6 signal quantification in 3 biological replicates , error bars indicate s . e . m . , normalized on SYBR ® - Gold signal ( Adj . Vol . ) , using Image lab software . * * * = p < 0 . 001 , * * = p < 0 . 01 , ns = not statistically significant , two - way ANOVA .

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C. Dot blot analysis of DNA:RNA hybrids formation: serial dilutions (750 ng, 375 ng, 187,5 ng, 93,75 ng, 46,87 ng) of genomic DNA extracted from WT or DIS3 silenced U2OS cells and pre-treated with RNase H, RNase III or mock treated, were arrayed on a membrane and probed using the S9.6 antibody. The histogram shows the S9.6 signal quantification in 3 biological replicates, error bars indicate s.e.m., normalized on SYBR®-Gold signal (Adj. Vol.), using Image lab software. *** = p < 0.001, ** = p < 0.01, ns= not statistically significant, two-way ANOVA.


D . DRIP - qPCR analysis at four different R - loops sites , RPL13A , ACTB , ENSA , and TRIM33 in U2OS cells transduced with shScr or shDIS3 and treated or not with RNase H . Results are shown as mean + s . e . m of three independent experiments . * * * * = p < 0 . 0001 , * * * = p < 0 . 001 , * = p < 0 . 05 , two - way ANOVA .

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D. DRIP-qPCR analysis at four different R-loops sites, RPL13A, ACTB, ENSA, and TRIM33 in U2OS cells transduced with shScr or shDIS3 and treated or not with RNase H. Results are shown as mean + s.e.m of three independent experiments.**** = p < 0.0001, *** = p < 0.001, * = p < 0.05, two-way ANOVA.


E . Representative pictures of comet assays performed under alkaline conditions in U2OS cells transfected for 48 hours with siCTRL or siDIS3 , untreated ( UNTR ) or treated with RNase H ( RNase H ) . Tail moments analysis was performed using Casplab software , error bars indicate s . e . m . Plots represent three biological replicates , with 150 cells for each cell line . * * * * = p < 0 . 0001 , ns = not statistically significant , two - way ANOVA , scale bar is 200 µm .

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E. Representative pictures of comet assays performed under alkaline conditions in U2OS cells transfected for 48 hours with siCTRL or siDIS3, untreated (UNTR) or treated with RNase H (RNase H). Tail moments analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with 150 cells for each cell line. **** = p < 0.0001, ns= not statistically significant, two-way ANOVA, scale bar is 200 µm.


A . U2OS cells were transfected with siCTRL or with siDIS3 and 48 hours later were irradiated ( IR ) or left untreated ( NO IR ) . Cells were fixed 4 hours after IR and stained for RAD51 . Histograms show foci number per cell , analyzed with Cell Profiler . The average number of foci per cell was calculated from three independent experiments . At least 200 cells were counted for each point , error bars indicate s . e . m . ( n = 3 ) . * = p < 0 . 05 , two - way ANOVA , ns = not statistically significant , scale bar is 10

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A. U2OS cells were transfected with siCTRL or with siDIS3 and 48 hours later were irradiated (IR) or left untreated (NO IR). Cells were fixed 4 hours after IR and stained for RAD51. Histograms show foci number per cell, analyzed with Cell Profiler. The average number of foci per cell was calculated from three independent experiments. At least 200 cells were counted for each point, error bars indicate s.e.m. (n=3). * = p < 0.05, two-way ANOVA, ns= not statistically significant, scale bar is 10 µ


D . Representative pictures of alkaline comet assays performed in U2OS silenced ( siDIS3 ) or not ( siCTRL ) for DIS3 , at sequential time points after exposure to 3 Gy irradiation . Relative comet tail moments were plotted for siCTRL ( black circles ) and siDIS3 ( red circles ) . Analysis was performed using Casplab software , error bars indicate s . e . m . Plots represent three biological replicates , with more than 150 cells for each siRNA . * * * * = p < 0 . 0001 , * * = p < 0 . 001 , ns = not statistically significant , two - way ANOVA , scale bar is 20 µm .

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D. Representative pictures of alkaline comet assays performed in U2OS silenced (siDIS3) or not (siCTRL) for DIS3, at sequential time points after exposure to 3 Gy irradiation. Relative comet tail moments were plotted for siCTRL (black circles) and siDIS3 (red circles). Analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with more than 150 cells for each siRNA. **** = p < 0.0001, ** = p < 0.001, ns= not statistically significant, two-way ANOVA, scale bar is 20 µm.


F . Representative images of HPRT negative mutants in HT1080 cells infected with scramble shRNA ( shScr ) or shRNA against DIS3 ( shDIS3 ) . Histograms show the mutation frequency and the deletion length of DIS3 WT ( shScr ) and DIS3 depleted cells ( shDIS3 ) after I - SceI induced DSB . Error bars represent s . e . m . ( n = 3 ) . * * * = p < 0 . 001 , ns = not statistically significant Student t - test .

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F. Representative images of HPRT negative mutants in HT1080 cells infected with scramble shRNA (shScr) or shRNA against DIS3 (shDIS3). Histograms show the mutation frequency and the deletion length of DIS3 WT (shScr) and DIS3 depleted cells (shDIS3) after I-SceI induced DSB. Error bars represent s.e.m. (n=3). *** = p < 0.001, ns= not statistically significant Student t-test.


A . U2OS cells were transfected with siCTRL or with siDIS3 and 48 hours later were 3 Gy irradiated ( IR ) or left untreated ( NO IR ) . Cells were fixed 4 hours after IR and stained for BRCA1 . Histograms show foci number per cell , analyzed with Cell Profiler . The average number of foci per cell was calculated from three independent experiments . At least 200 cells were counted for each point , error bars indicate s . e . m . ( n = 3 biological replicates ) . * * = p < 0 . 01 , ns = not statistically significant , two - way ANOVA , scale bar is 10 µm .

A. U2OS cells were transfected with siCTRL or with siDIS3 and 48 hours later were 3 Gy irradiated (IR) or left untreated (NO IR). Cells were fixed 4 hours after IR and stained for BRCA1. Histograms show foci number per cell, analyzed with Cell Profiler. The average number of foci per cell was calculated from three independent experiments. At least 200 cells were counted for each point, error bars indicate s.e.m. (n=3 biological replicates). ** = p < 0.01, ns= not statistically significant, two-way ANOVA, scale bar is 10 µm.


C . Representative images of BRCA1 staining on U2OS cells transfected with siCTRL or with siDIS3 . 48 hours after transfection cells were irradiated and 4 hours later were treated with RNase H or left untreated ( UNTR ) . Histograms show the average foci number per cell from three independent experiments analyzed with Cell Profiler . At least 200 cells were counted for each point , error bars indicate s . e . m . ( n = 3 ) . * * = p < 0 . 01 , ns = not statistically significant , one - way ANOVA , scale bar is 10 µm .

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C. Representative images of BRCA1 staining on U2OS cells transfected with siCTRL or with siDIS3. 48 hours after transfection cells were irradiated and 4 hours later were treated with RNase H or left untreated (UNTR). Histograms show the average foci number per cell from three independent experiments analyzed with Cell Profiler. At least 200 cells were counted for each point, error bars indicate s.e.m. (n=3). ** = p < 0.01, ns= not statistically significant, one-way ANOVA, scale bar is 10 µm.


D . Representative pictures of comet assays performed under alkaline conditions in U2OS cells , silenced ( siDIS3 ) or not ( siCTRL ) for DIS3 untreated ( UNTR ) or treated with RNase H ( RNase H ) . Relative comet tail moments are plotted for siCTRL ( black circles ) and siDIS3 ( red circles ) . Analysis was performed using Casplab software , error bars indicate s . e . m . Plots represent three biological replicates , with 150 cells for each siRNA . * * * * = p < 0 . 0001 , one - way ANOVA , scale bar is 20 µm .

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D. Representative pictures of comet assays performed under alkaline conditions in U2OS cells, silenced (siDIS3) or not (siCTRL) for DIS3 untreated (UNTR) or treated with RNase H (RNase H). Relative comet tail moments are plotted for siCTRL (black circles) and siDIS3 (red circles). Analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with 150 cells for each siRNA. **** = p < 0.0001, one-way ANOVA, scale bar is 20 µm.


A . Representative confocal images upon irradiation with the 405nm laser . U2OS cells were presensitized with BrdU for 24 hours , micro - irradiated , fixed after 10 minutes , and stained with antibodies against γH2AX and DIS3 scale bar is 10 µm . B . Magnification image of the white box in panel A . C . Intensity profiles of γH2AX ( green ) and DIS3 signals through the yellow line in panel B . Quantification is performed with ImageJ software scale bar is 10 µm .

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A. Representative confocal images upon irradiation with the 405nm laser. U2OS cells were presensitized with BrdU for 24 hours, micro-irradiated, fixed after 10 minutes, and stained with antibodies against γH2AX and DIS3 scale bar is 10 µm. B. Magnification image of the white box in panel A. C. Intensity profiles of γH2AX (green) and DIS3 signals through the yellow line in panel B. Quantification is performed with ImageJ software scale bar is 10 µm.


A . Representative pictures of comet assays performed under alkaline conditions in DIS3 WT MM cell lines MM . 1S and RPMI - 8226 ( RPMI ) , and in DIS3 mutated MM cell lines PCM6 and OPM2 , untreated ( UNTR ) or treated with RNase H ( RNase H ) . Tail moments analysis was performed using Casplab software , error bars indicate s . e . m . Plots represent three biological replicates , with 150 cells for each cell line . * * * * = p < 0 . 0001 ns = not statistically significant , two - way ANOVA , scale bar is 20 µm .

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A. Representative pictures of comet assays performed under alkaline conditions in DIS3 WT MM cell lines MM.1S and RPMI-8226 (RPMI), and in DIS3 mutated MM cell lines PCM6 and OPM2, untreated (UNTR) or treated with RNase H (RNase H). Tail moments analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with 150 cells for each cell line. **** = p < 0.0001 ns= not statistically significant, two-way ANOVA, scale bar is 20 µm.


B . Immunostaining with S9 . 6 ( green ) and DAPI ( blue ) in MM . 1S , RPMI - 8226 , PCM6 , and OPM2 cells , treated ( RNase H ) or not ( UNTR ) with RNase H for 1 hour . Histograms show S9 . 6 signal intensity per cell . More than 150 cells were counted , error bars indicate s . e . m . ( n = 3 biological replicates ) . * * * * = p < 0 . 0001 , one - way ANOVA , scale bar is 10 µm .

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B. Immunostaining with S9.6 (green) and DAPI (blue) in MM.1S, RPMI-8226, PCM6, and OPM2 cells, treated (RNase H) or not (UNTR) with RNase H for 1 hour. Histograms show S9.6 signal intensity per cell. More than 150 cells were counted, error bars indicate s.e.m. (n=3 biological replicates). **** = p < 0.0001, one-way ANOVA, scale bar is 10 µm.


A . Relative mRNA expression level of interferon genes IFI6 , ISG15 , IFITM1 , IFIT3 , IFI27 , MCL1 , cGAS , and STING in OPM2 cells infected with a vector expressing DIS3 ( DIS3 ) or an empty vector ( EV ) . Values are relative to housekeeping gene expression , normalized on EV , and represent SE of three biological replicates . * * * = p < 0 . 001 , * * = p < 0 . 01 , multiple t - tests .

A. Relative mRNA expression level of interferon genes IFI6, ISG15, IFITM1, IFIT3, IFI27, MCL1, cGAS, and STING in OPM2 cells infected with a vector expressing DIS3 (DIS3) or an empty vector (EV). Values are relative to housekeeping gene expression, normalized on EV, and represent SE of three biological replicates. *** = p < 0.001, ** = p < 0.01, multiple t-tests.


Differentially expressed genes ( DEGs ) between mutant and WT samples ; p - values are from limma and false - discovery rate ( FDR ) is calculated using the Benjamini - Hochberg procedure ; FDR < 0 . 05 was used as cut off . Functional annotation analysis by IPA of the DEGs between DIS3 mutant and WT samples based on FDR cut - off . ( C ) Volcano plot of DEGs between mutant and WT samples ; labeled genes are the IFN - related genes with an FDR < 0 . 05 ( red horizontal line ) and absolute log 2 - fold change above or below 0 . 5 ( red vertical lines ) , respectively ; additional representative IFN - related genes are also labeled .

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Differentially expressed genes (DEGs) between mutant and WT samples; p-values are from limma and false-discovery rate (FDR) is calculated using the Benjamini-Hochberg procedure; FDR < 0.05 was used as cut off. Functional annotation analysis by IPA of the DEGs between DIS3 mutant and WT samples based on FDR cut-off. (C) Volcano plot of DEGs between mutant and WT samples; labeled genes are the IFN-related genes with an FDR < 0.05 (red horizontal line) and absolute log 2-fold change above or below 0.5 (red vertical lines), respectively; additional representative IFN-related genes are also labeled.


( F ) Violin plot of the number of non - synonymous ( NS ) mutation load comparison between DIS3 mutant versus DIS3 WT performed on the entire CoMMpass dataset , including 853 MM patients , 88 presenting DIS3 mutations ( all samples ) and on the CoMMpass dataset , stratified according to the intensity of the IFN responsiveness ( clusters C1 , C2 , C3 ) . Wilcoxon rank - sum test was used for comparison between DIS3 mutant vs WT . * * * * = p < , * * = p < 0 . 01 , * = p < 0 . 05 . Black color dots represent the median in each group .

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(F) Violin plot of the number of non-synonymous (NS) mutation load comparison between DIS3 mutant versus DIS3 WT performed on the entire CoMMpass dataset, including 853 MM patients, 88 presenting DIS3 mutations (all samples) and on the CoMMpass dataset, stratified according to the intensity of the IFN responsiveness (clusters C1, C2, C3). Wilcoxon rank-sum test was used for comparison between DIS3 mutant vs WT. **** = p <, ** = p < 0.01, * = p < 0.05. Black color dots represent the median in each group.


A - E Chemokine - galectin interactions were detected by using a solid - phase immunoassay . For this , 46 human CC and CXC chemokines were adsorbed on nitrocellulose membranes and the stripes incubated in parallel with ( A ) TBS or ( B ) TBS containing biotinylated galectins ( the representative image shows a processed membrane tested with labeled Gal - 3 ) . Signals had been generated by using SA - HRP and chemiluminescence reagents . ( C - D ) The blots were subjected to densitometric analysis , and ( E ) all independent experiments were combined ( binding chemokines in light blue , Gal - 3 : n = 5 , Gal - 3 CRD : n = 5 , Gal - 1 n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Chemokine-galectin interactions were detected by using a solid-phase immunoassay. For this, 46 human CC and CXC chemokines were adsorbed on nitrocellulose membranes and the stripes incubated in parallel with (A) TBS or (B) TBS containing biotinylated galectins (the representative image shows a processed membrane tested with labeled Gal-3). Signals had been generated by using SA-HRP and chemiluminescence reagents. (C-D) The blots were subjected to densitometric analysis, and (E) all independent experiments were combined (binding chemokines in light blue, Gal-3: n = 5, Gal-3 CRD: n = 5, Gal-1 n = 4).


For SPR - based experiments , ( F ) Gal - 3 ( density 650 RU ) , were immobilized and increasing concentrations of CXCL12 were passed over the flow cells . The red curve represents a single - site fit to the data . Insets are representative sensorgrams of CXCL12 testing on immobilized galectin .

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

For SPR-based experiments, (F) Gal-3 (density 650 RU), were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.


For SPR - based experiments , ( G ) Gal - 3 CRD ( density 1180 RU ) , were immobilized and increasing concentrations of CXCL12 were passed over the flow cells . The red curve represents a single - site fit to the data . Insets are representative sensorgrams of CXCL12 testing on immobilized galectin .

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

For SPR-based experiments, (G) Gal-3 CRD (density 1180 RU), were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.


For SPR - based experiments , ( H ) Gal - 1 ( density 130 RU ) were immobilized and increasing concentrations of CXCL12 were passed over the flow cells . The red curve represents a single - site fit to the data . Insets are representative sensorgrams of CXCL12 testing on immobilized galectin .

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

For SPR-based experiments, (H) Gal-1 (density 130 RU) were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.


a - b Expansions of 1H - 15N HSQC spectra are overlaid and Δδ values are plotted vs . the amino acid sequences , along with secondary structures : ( a ) 10 µM 15N - enriched CXCL12 alone ( black peaks ) and in the presence of 330 µM Gal - 3 CRD ( red peaks ; assignments reported by Murphy et al . [ 74 ] ) and ( b ) 30 µM 15N - enriched Gal - 3 CRD alone ( black peaks ) and in the presence of 500 µM CXCL12 ( red peaks ; assignments for Gal -

a-b Expansions of 1H-15N HSQC spectra are overlaid and Δδ values are plotted vs. the amino acid sequences, along with secondary structures: (a) 10 µM 15N-enriched CXCL12 alone (black peaks) and in the presence of 330 µM Gal-3 CRD (red peaks; assignments reported by Murphy et al.[74]) and (b) 30 µM 15N-enriched Gal-3 CRD alone (black peaks) and in the presence of 500 µM CXCL12 (red peaks; assignments for Gal-3


a - c Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of ( a ) Gal - 3 , ( b ) Gal - 3 CRD , and ( c ) Gal - 1 ( A - C : n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-c Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of (a) Gal-3, (b) Gal-3 CRD, and (c) Gal-1 (A-C: n = 4).


D Human CD4 + T cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal - 3 ( dark green ) , Gal - 3 CRD ( light green ) , and Gal - 1 ( blue , n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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 Human CD4+ T cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3 (dark green), Gal-3 CRD (light green), and Gal-1 (blue, n = 3).


E - F ( E ) Jurkat T cells migrated to increasing concentrations of CXCL12 alone ( black ) and CXCL12 in the presence of 0 . 1 nM of Gal - 3 CRD ( light green ) . ( F ) The inhibitory effect of Gal - 3 CRD is shown as percentage of the chemotactic effect of CXCL12 ( E - F : n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (E) Jurkat T cells migrated to increasing concentrations of CXCL12 alone (black) and CXCL12 in the presence of 0.1 nM of Gal-3 CRD (light green). (F) The inhibitory effect of Gal-3 CRD is shown as percentage of the chemotactic effect of CXCL12 (E-F: n = 3).


G Jurkat T cells were allowed to migrate in the presence of CXCL12 at increasing concentrations alone and with 0 . 1 nM Gal - 1 or 100 nM Gal - 1 ( n = 3 ) .

O O O O O O O O O O O O O O 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 Jurkat T cells were allowed to migrate in the presence of CXCL12 at increasing concentrations alone and with 0.1 nM Gal-1 or 100 nM Gal-1 (n=3).


H Jurkat T cells did not migrate in the sole presence of 1 nM to 1 µM Gal - 3 , Gal - 3 CRD ( both n = 5 ) , or Gal - 1 ( n = 4 ) .

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

H Jurkat T cells did not migrate in the sole presence of 1 nM to 1 µM Gal-3, Gal-3 CRD (both n = 5), or Gal-1 (n = 4).


I The viability of Jurkat T cells was assessed after incubation with 1 µM CXCL12 alone and in the presence of Gal - 3 , Gal - 3 CRD and Gal - 1 ( n = 2 ) .

O O O O O O O O O O O O O O O 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 The viability of Jurkat T cells was assessed after incubation with 1 µM CXCL12 alone and in the presence of Gal-3, Gal-3 CRD and Gal-1 (n = 2).


( J ) THP - 1 cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal - 3 , Gal - 3 CRD and Gal -

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(J) THP-1 cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3, Gal-3 CRD and Gal-1


( K ) neutrophils migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal - 3 , Gal - 3 CRD and Gal -

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(K) neutrophils migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3, Gal-3 CRD and Gal-1


L Human eosinophils migrated in the presence of 10 nM CCL26 alone and with 10 nM Gal - 3 , Gal - 3 CRD , and Gal - 1 ( n = 3 ) .

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L Human eosinophils migrated in the presence of 10 nM CCL26 alone and with 10 nM Gal-3, Gal-3 CRD, and Gal-1 (n = 3).


M CD4 + T cells migrated in the presence of 1 nM CCL17 alone and with equimolar concentrations of Gal - 3 , Gal - 3 CRD , and Gal - 1 ( n = 4 ) .

M CD4+ T cells migrated in the presence of 1 nM CCL17 alone and with equimolar concentrations of Gal-3, Gal-3 CRD, and Gal-1 (n = 4).


A - B The peritoneal recruitment of ( A ) CD45 + / CD115 - / Ly6G + neutrophils and ( B ) CD45 + / CD115 + / Ly6Chi classical monocytes in C57BL / 6J mice was assessed 4 hours after intraperitoneal ( IP ) injection of 500 nM CXCL12 alone ( black ) and in the presence of 50 nM Gal - 3 ( dark green ; A - B : n = 7 mice ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The peritoneal recruitment of (A) CD45+/CD115-/Ly6G+ neutrophils and (B) CD45+/CD115+/Ly6Chi classical monocytes in C57BL/6J mice was assessed 4 hours after intraperitoneal (IP) injection of 500 nM CXCL12 alone (black) and in the presence of 50 nM Gal-3 (dark green; A-B: n = 7 mice).


C The peritoneal recruitment of classical monocytes after IP injection of PBS ( n = 6 ) or TG in C57BL / 6J WT ( dark green , n = 10 mice ) and Gal - 3 - / - ( black , n = 5 mice ) mice was assessed after 18 h . Where indicated , the mice received an IP injection of CXCR4 antagonist AMD 3465 12 hours prior to the experiment .

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C The peritoneal recruitment of classical monocytes after IP injection of PBS (n = 6) or TG in C57BL/6J WT (dark green, n = 10 mice) and Gal-3-/- (black, n = 5 mice) mice was assessed after 18 h. Where indicated, the mice received an IP injection of CXCR4 antagonist AMD 3465 12 hours prior to the experiment.


D The concentration of CXCL12 concentration was determined by ELISA on the peritoneal lavage normalized with levels from the mesenterium ( n = 4 mice ) .

O O O 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 The concentration of CXCL12 concentration was determined by ELISA on the peritoneal lavage normalized with levels from the mesenterium (n = 4 mice).


E CXCR4 expression levels on Ly6Chi monocytes of the blood and the peritoneal lavage after 18 h of TG stimulation were determined by flow cytometry and indicated as mean fluorescence intensity ( MFI ) ( n = 4 mice ) .

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E CXCR4 expression levels on Ly6Chi monocytes of the blood and the peritoneal lavage after 18 h of TG stimulation were determined by flow cytometry and indicated as mean fluorescence intensity (MFI) (n = 4 mice).


A - B Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of E185A ( orange , n = 4 ) and N222A ( red , n = 5 ) mutants of ( A ) Gal - 3 and ( B ) Gal - 3 CRD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of E185A (orange, n = 4) and N222A (red, n = 5) mutants of (A) Gal-3 and (B) Gal-3 CRD.


C - D Jurkat T cells migrated in the presence of 10 nM CXCL12 with 10 nM ( C ) Gal - 3 and ( D ) Gal - 3 CRD mutants as indicated ( C - D : n = 3 ) .

O O O O O O O O O O O O O O O O O O O O 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 Jurkat T cells migrated in the presence of 10 nM CXCL12 with 10 nM (C) Gal-3 and (D) Gal-3 CRD mutants as indicated (C-D: n = 3).


E Aggregation of Jurkat T cells in the presence of 1 nM to 1 µM Gal - 3 and Gal - 1 alone and with 70 mM lactose , and Gal - 3 CRD , was determined by flow cytometry ( n = 4 ) .

O O O 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

E Aggregation of Jurkat T cells in the presence of 1 nM to 1 µM Gal-3 and Gal-1 alone and with 70 mM lactose, and Gal-3 CRD, was determined by flow cytometry (n = 4).


Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of ( F ) 1 µM Gal - 3 , ( G ) Gal - 1 , ( H ) Gal - 3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ .

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

Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of (F) 1 µM Gal-3, (G) Gal-1, (H) Gal-3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ.


Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of ( I ) 0 . 1 nM Gal - 3 and ( J ) Gal - 3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ .

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

Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of (I) 0.1 nM Gal-3 and (J) Gal-3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ.


a HEK cells transfected with a luminescent cAMP sensor were incubated with 10 nM CXCL12 alone and in the presence of 10 nM Gal - 3 CRD followed by stimulation with forskolin , an activator of adenylate cyclase . Results are shown as luminescence relative to baseline ( RLU , example of n = 3 ) .

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a HEK cells transfected with a luminescent cAMP sensor were incubated with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD followed by stimulation with forskolin, an activator of adenylate cyclase. Results are shown as luminescence relative to baseline (RLU, example of n = 3).


b The effect of 10 nM Gal - 3 CRD upon stimulation with 10 nM CXCL12 prior to forskolin stimulation , was tested . Control experiments were performed as indicated ( n = 3 , four technical replicates ) .

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

b The effect of 10 nM Gal-3 CRD upon stimulation with 10 nM CXCL12 prior to forskolin stimulation, was tested. Control experiments were performed as indicated (n = 3, four technical replicates).


c HEK cells transfected with a RlucII - conjugated CXCR4 and an eYFP - β - arrestin 2 construct were stimulated with 10 nM CXCL12 alone and in the presence of 10 nM Gal - 3 CRD . Control experiments were performed as indicated . Results are given as the net BRET ratio ( i . e . ratio of emissions at 535 / 485 nm minus the ratio of mock cells , n = 5 , three technical replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK cells transfected with a RlucII-conjugated CXCR4 and an eYFP-β-arrestin 2 construct were stimulated with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD. Control experiments were performed as indicated. Results are given as the net BRET ratio (i.e. ratio of emissions at 535/485 nm minus the ratio of mock cells, n = 5, three technical replicates).


D Internalization of CXCR4 with 10 nM CXCL12 alone and in the presence of 10 nM Gal - 3 CRD was assessed by incubation with an APC - conjugated anti - CXCR4 antibody ( baseline signal = 100 % , n = 8 , two technical replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 Internalization of CXCR4 with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD was assessed by incubation with an APC-conjugated anti-CXCR4 antibody (baseline signal = 100%, n = 8, two technical replicates).


E 100 nM CXCL12 and Gal - 3 were added to Jurkat T cells , and their co - localization was assessed on the cell surface by PLA . Control experiments were performed as indicated ( representative example of n = 3 ) . White scale bar : 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 100 nM CXCL12 and Gal-3 were added to Jurkat T cells, and their co-localization was assessed on the cell surface by PLA. Control experiments were performed as indicated (representative example of n = 3). White scale bar: 10 µm.


F - G Jurkat T cells were incubated with 100 nM of Gal - 3 CRD Alexa Fluor 555 alone , with 10 µM AMD 3100 , with 100 nM CXCL12 or with CXCL12 and AMD 3100 . Experiments were performed ( f ) without and ( g ) in the presence of 70 mM lactose ( F - G : n = 3 , two technical replicates ) .

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

F-G Jurkat T cells were incubated with 100 nM of Gal-3 CRD Alexa Fluor 555 alone, with 10 µM AMD 3100, with 100 nM CXCL12 or with CXCL12 and AMD 3100. Experiments were performed (f) without and (g) in the presence of 70 mM lactose (F-G: n = 3, two technical replicates).


H Jurkat T cells were incubated with 100 nM CXCL12 FITC alone and with increasing concentrations of Gal - 3 CRD as indicated ( n = 4 , two technical replicates ) .

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

H Jurkat T cells were incubated with 100 nM CXCL12 FITC alone and with increasing concentrations of Gal-3 CRD as indicated (n = 4, two technical replicates).


ISCts midguts expressing YFP alone ( control ) , or expressing two non - overlapping EcR - RNAi , and quantification of the number of YFP - positive cells ( yellow ) . The graph also plots the number of GFP - positive cells in esgts midgut expressing GFP alone ( ctrl ) , or expressing EcR - RNAi # 1 , EcR - RNAi # 2 , and mcherry - RNAi as an additional negative control ( see EV2B ) .

ISCts midguts expressing YFP alone (control), or expressing two non-overlapping EcR-RNAi, and quantification of the number of YFP-positive cells (yellow). The graph also plots the number of GFP-positive cells in esgts midgut expressing GFP alone (ctrl), or expressing EcR-RNAi#1, EcR-RNAi#2, and mcherry-RNAi as an additional negative control (see EV2B).


esgts midguts expressing GFP alone ( control ) , or expressing EcR - DN , and EcR - DN + pri . Samples were stained for GFP ( green ) . The graph shows quantification of the number of GFP - positive cells in the different genotypes .

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

esgts midguts expressing GFP alone (control), or expressing EcR-DN, and EcR-DN + pri. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in the different genotypes.


Posterior midguts containing control and Ubr3 null MARCM clones ( GFP , green ) .

Posterior midguts containing control and Ubr3 null MARCM clones (GFP, green).


esgts midguts expressing GFP alone ( control ) , or expressing Ubr3 - RNAi , and Ubr3 - RNAi + OvoB . Samples were stained for GFP ( green ) . H ' quantification of GFP - positive cells from H .

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

esgts midguts expressing GFP alone (control), or expressing Ubr3-RNAi, and Ubr3-RNAi + OvoB. Samples were stained for GFP (green). H' quantification of GFP-positive cells from H.


esgts midguts expressing GFP alone ( control ) or expressing SvbACT . Samples were stained for GFP ( green ) , and ß - catenin ( purple ) , DE - Cadherin ( white ) or Scribble ( yellow ) . Top and bottom pictures show separate channels for a same region .

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

esgts midguts expressing GFP alone (control) or expressing SvbACT. Samples were stained for GFP (green), and ß-catenin (purple), DE-Cadherin (white) or Scribble (yellow). Top and bottom pictures show separate channels for a same region.


esgts midguts expressing GFP alone ( control ) , or expressing miR8 , OvoB , and mir8 + OvoB . Samples were stained for GFP ( green ) and ß - catenin ( purple ) . The graph shows quantification of GFP - positive cells for each genotype . The Y axis is drawn using a log ( 10 ) scale .

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

esgts midguts expressing GFP alone (control), or expressing miR8, OvoB, and mir8 + OvoB. Samples were stained for GFP (green) and ß-catenin (purple). The graph shows quantification of GFP-positive cells for each genotype. The Y axis is drawn using a log(10) scale.


esgts midguts expressing GFP alone ( control ) , or expressing Notch Intra Cellular Domain ( NICD ) , and NICD + OvoB . In closeups ( right ) , DAPI is shown in purple for improved contrast ; the arrow highlights a cell with intermediate phenotype .

esgts midguts expressing GFP alone (control), or expressing Notch Intra Cellular Domain (NICD), and NICD + OvoB. In closeups (right), DAPI is shown in purple for improved contrast; the arrow highlights a cell with intermediate phenotype.


esgtsF / O midguts expressing GFP alone ( control ) , or expressing RasV12 , and RasV12 + svb - RNAi ( top panels ) . Bottom panels show esgts midguts expressing GFP alone ( control ) , or expressing EGFR - DN , and EGFR - DN + OvoB . Samples were stained for GFP ( green ) . The graph shows quantification of the number of GFP - positive cells in esgts midguts expressing GFP alone ( ctrl ) , or expressing EGFR - DN , EGFR - DN + OvoB , and EGFR - DN + svb - RNAi . The Y axis is plotted as log ( 10 ) .

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esgtsF/O midguts expressing GFP alone (control), or expressing RasV12, and RasV12 + svb-RNAi (top panels). Bottom panels show esgts midguts expressing GFP alone (control), or expressing EGFR-DN, and EGFR-DN + OvoB. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in esgts midguts expressing GFP alone (ctrl), or expressing EGFR-DN, EGFR-DN + OvoB, and EGFR-DN + svb-RNAi. The Y axis is plotted as log(10).


esgts midguts expressing GFP alone ( control ) , or expressing ArmS10 , ArmS10 + svb - RNAi , ArmS10 + OvoB , TCF - DN , and TCF - DN + OvoB . Samples were stained for GFP ( green ) . The graph shows quantification of the number of GFP - positive cells in esgts midguts expressing GFP alone ( ctrl ) , or expressing TCF - DN , TCF - DN + OvoB , and TCF - DN + svb - RNAi . The Y axis is plotted as log ( 10 ) .

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esgts midguts expressing GFP alone (control), or expressing ArmS10, ArmS10 + svb-RNAi, ArmS10 + OvoB ,TCF-DN, and TCF-DN + OvoB. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in esgts midguts expressing GFP alone (ctrl), or expressing TCF-DN, TCF-DN + OvoB, and TCF-DN + svb-RNAi. The Y axis is plotted as log(10).


esgts midguts expressing GFP alone ( control ) , or expressing SvbREP . Samples were stained for GFP ( green ) and Prospero ( red ) . Closeups correspond to boxed regions , with DAPI shown in purple and GFP - positive cells outlined in yellow .

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

esgts midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and Prospero (red). Closeups correspond to boxed regions, with DAPI shown in purple and GFP-positive cells outlined in yellow.


esgts - F / O midguts expressing GFP alone ( control ) , or expressing SvbREP . Samples were stained for GFP ( green ) and cleaved DCP1 ( red ) . E ' shows quantification of the number of GFP - positive cells in esgts guts expressing GFP alone ( ctrl ) , or DIAP , SvbREP , and SvbREP + DIAP .

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esgts-F/O midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and cleaved DCP1 (red). E' shows quantification of the number of GFP-positive cells in esgts guts expressing GFP alone (ctrl), or DIAP, SvbREP, and SvbREP + DIAP.


esgts midguts expressing GFP alone ( control ) , or expressing Notch - RNAi , Notch - RNAi + svb - RNAi , and Notch - RNAi + SvbREP . Samples were stained for GFP ( green ) and Prospero ( red ) . The graph shows quantification of the number of GFP - positive cells .

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esgts midguts expressing GFP alone (control), or expressing Notch-RNAi, Notch-RNAi + svb-RNAi, and Notch-RNAi + SvbREP. Samples were stained for GFP (green) and Prospero (red). The graph shows quantification of the number of GFP-positive cells.


esgts midguts expressing GFP alone ( control ) , or expressing STAT - RNAi , STAT - RNAi + svb - RNAi and STAT - RNAi + SvbREP . Samples were stained for GFP ( green ) and Prospero ( red ) . The graph shows quantification of the number of GFP - positive cells .

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esgts midguts expressing GFP alone (control), or expressing STAT-RNAi, STAT-RNAi + svb-RNAi and STAT-RNAi + SvbREP. Samples were stained for GFP (green) and Prospero (red). The graph shows quantification of the number of GFP-positive cells.


Cross sections of control MyoIAts midguts ( expressing GFP and mCherry - RNAi ) , or expressing svb - RNAi , SvbACT , and Pri . Samples were stained for F - actin ( white ) , GFP ( green ) and Tsp2a ( yellow ) .

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Cross sections of control MyoIAts midguts (expressing GFP and mCherry-RNAi), or expressing svb-RNAi, SvbACT, and Pri. Samples were stained for F-actin (white), GFP (green) and Tsp2a (yellow).


MyoIAts midguts expressing GFP alone ( control ) , or expressing SvbACT . Samples were stained for GFP ( green ) , Scribble ( yellow ) , ß - catenin ( purple ) and DAPI ( Blue ) . A ' pictures display cross sections of the regions shown in A .

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MyoIAts midguts expressing GFP alone (control), or expressing SvbACT. Samples were stained for GFP (green), Scribble (yellow), ß-catenin (purple) and DAPI (Blue). A' pictures display cross sections of the regions shown in A.


MyoIAts midguts expressing GFP alone ( control ) , or expressing SvbACT . Samples were stained for GFP ( green ) and PH3 ( red ) . The graph plots number of mitotic PH3 - positive cells per midgut of MyoIAts guts expressing GFP alone ( ctrl ) , or expressing SvbACT , and SvbREP + pri .

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MyoIAts midguts expressing GFP alone (control), or expressing SvbACT. Samples were stained for GFP (green) and PH3 (red). The graph plots number of mitotic PH3-positive cells per midgut of MyoIAts guts expressing GFP alone (ctrl), or expressing SvbACT , and SvbREP + pri.


( D ) Cells were treated with sodium arsenite ( 50 µM ) for 45 min , followed by recovery in drug - free medium . Kinetics of SG dissolution are reported . Images were taken over a time period of 4 hrs every 10 min . Dashed lines = 95 % confidence intervals . Number of cells counted : 159 ( siRNA control ) ; 166 ( siRNA Hsp90 α + β ) .

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(D) Cells were treated with sodium arsenite (50 µM) for 45 min, followed by recovery in drug-free medium. Kinetics of SG dissolution are reported. Images were taken over a time period of 4 hrs every 10 min. Dashed lines = 95% confidence intervals. Number of cells counted: 159 (siRNA control); 166 (siRNA Hsp90 α+β).


( E ) HeLa - Kyoto cells were treated with HS at 43 . 5 ˚ C for 1 hr . Cells were then allowed to recover at 37 ° C for 1 hr in drug - free medium ( control ) or in presence of VER ( 40 µM ) , GA ( 5 µM ) or 17AAG ( 5 µM ) . Cells were fixed , stained for the SG marker TIA - 1 and the percentage of cells with SGs was counted . Number of cells counted : 605 ( recovery control ) ; 510 ( recovery VER ) ; 1334 ( recovery GA ) ; 468 ( recovery 17AAG ) . n = 3 - 4 independent experiments , ± sem . p < 0 . 01 ( One - way ANOVA ) . ( F ) G3BP2 - GFP HeLa - Kyoto cells were treated with MG132 ( 20 µM ) for 3 hrs , followed by recovery for 2 hrs in drug - free medium ( control ) or in presence of VER ( 40 µM ) , GA ( 5 µM ) or 17AAG ( 5 µM ) . Cells were fixed and the percentage of cells with SGs was counted . Number of cells counted : 439 ( recovery control ) ; 649 ( recovery VER ) ; 637 ( recovery GA ) ; 649 ( recovery 17AAG ) . n = 3 independent experiments , ± sem . p < 0 . 0001 ( One - way ANOVA ) .

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(E) HeLa-Kyoto cells were treated with HS at 43.5˚C for 1 hr. Cells were then allowed to recover at 37 °C for 1 hr in drug-free medium (control) or in presence of VER (40 µM), GA (5 µM) or 17AAG (5 µM). Cells were fixed, stained for the SG marker TIA-1 and the percentage of cells with SGs was counted. Number of cells counted: 605 (recovery control); 510 (recovery VER); 1334 (recovery GA); 468 (recovery 17AAG). n = 3-4 independent experiments, ± sem. p < 0.01 (One-way ANOVA). (F) G3BP2-GFP HeLa-Kyoto cells were treated with MG132 (20 µM) for 3 hrs, followed by recovery for 2 hrs in drug-free medium (control) or in presence of VER (40 µM), GA (5 µM) or 17AAG (5 µM). Cells were fixed and the percentage of cells with SGs was counted. Number of cells counted: 439 (recovery control); 649 (recovery VER); 637 (recovery GA); 649 (recovery 17AAG). n = 3 independent experiments, ± sem. p < 0.0001 (One-way ANOVA).


( C ) HeLa cells were subjected to proximity ligation assay ( PLA ) using antibodies specific for endogenous Hsp90 and DYRK3 . PLA foci and nuclei were segmented , and PLA foci / cell were automatically quantified . The mean of PLA / foci in cells incubated with no antibodies ( - ) , only Hsp90 antibody , only DYRK3 antibody and both Hsp90 and DYRK3 antibodies is shown . Cells incubated with Hsp90 and DYRK3 antibodies and left untreated were used as control . n = 3 independent experiments , ± sem ; 368 - 504 cells counted / sample , p < 0 . 002 between the negative controls and cells incubated with Hsp90 and DYRK3 antibodies ; p = 10 - 6 between cells incubated with Hsp90 and DYRK3 antibodies and left untreated ( control ) or treated for 2 hrs with GA ( 5 µM ) ( One - way ANOVA ) . Scale bar is 10 µm .

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(C) HeLa cells were subjected to proximity ligation assay (PLA) using antibodies specific for endogenous Hsp90 and DYRK3. PLA foci and nuclei were segmented, and PLA foci/cell were automatically quantified. The mean of PLA/foci in cells incubated with no antibodies (-), only Hsp90 antibody, only DYRK3 antibody and both Hsp90 and DYRK3 antibodies is shown. Cells incubated with Hsp90 and DYRK3 antibodies and left untreated were used as control. n = 3 independent experiments, ± sem; 368-504 cells counted/sample, p < 0.002 between the negative controls and cells incubated with Hsp90 and DYRK3 antibodies; p = 10-6 between cells incubated with Hsp90 and DYRK3 antibodies and left untreated (control) or treated for 2 hrs with GA (5 µM) (One-way ANOVA). Scale bar is 10 µm.


( F ) HeLa cells were lipofected with a cDNA encoding for GFP or GFP - DYRK3 . 6 hrs post - transfection , cells were incubated in drug - free medium ( - ) or in presence of GA , 17AAG or VER ( concentrations are shown ) . Total proteins were extracted 16 hrs later . GFP and TUBA4A protein levels were analyzed by immunoblotting . Representative immunoblotting of 3 independent experiments .

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(F) HeLa cells were lipofected with a cDNA encoding for GFP or GFP-DYRK3. 6 hrs post-transfection, cells were incubated in drug-free medium (-) or in presence of GA, 17AAG or VER (concentrations are shown). Total proteins were extracted 16 hrs later. GFP and TUBA4A protein levels were analyzed by immunoblotting. Representative immunoblotting of 3 independent experiments.


( I ) HeLa cells were lipofected with a cDNA encoding for GFP - DYRK3 . 24 hrs post - transfection , cells were treated for 45 min with arsenite ( 50 µM ) alone ( control ) or with GA ( 5 µM ) or 17AAG ( 5 µM ) . Cell were then fixed and the percentage of cells expressing GFP - DYRK3 with SGs was counted . Total cells counted : 462 ( sodium arsenite ) ; 444 ( sodium arsenite and GA ) ; 561 ( sodium arsenite and 17AAG ) . n = 3 independent experiments , ± sem ; p < 0 . 001 ( One - way ANOVA ) .

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(I) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3. 24 hrs post-transfection, cells were treated for 45 min with arsenite (50 µM) alone (control) or with GA (5 µM) or 17AAG (5 µM). Cell were then fixed and the percentage of cells expressing GFP-DYRK3 with SGs was counted. Total cells counted: 462 (sodium arsenite); 444 (sodium arsenite and GA); 561 (sodium arsenite and 17AAG). n = 3 independent experiments, ± sem; p < 0.001 (One-way ANOVA).


( A , HeLa cells were left untreated ( control ) or treated with GA ( 5 µM ) , 17AAG ( 5 µM ) or VER ( 10 µM ) for 8 hrs . Cells were then fixed and immunostained for the nuclear speckle marker SC35 and DYRK3 . Automatic segmentation of nuclear speckles is based on SC35 . An automated imaging assay was used to quantify the percentage of nuclear speckles highly enriched for DYRK3 ( using a fluorescent ratio 1 . 5 as threshold ) . Scale bar is 10 µm .

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(A, HeLa cells were left untreated (control) or treated with GA (5 µM), 17AAG (5 µM) or VER (10 µM) for 8 hrs. Cells were then fixed and immunostained for the nuclear speckle marker SC35 and DYRK3. Automatic segmentation of nuclear speckles is based on SC35. An automated imaging assay was used to quantify the percentage of nuclear speckles highly enriched for DYRK3 (using a fluorescent ratio 1.5 as threshold). Scale bar is 10 µm.


D ) Representative confocal microscopy images of cells with SC35 - positive bodies ( D ) Scale bar is 10 µm .

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D) Representative confocal microscopy images of cells with SC35-positive bodies (D) Scale bar is 10 µm.


( F ) HeLa cells were lipofected with a cDNA encoding for GFP - DYRK3 - dN . 24 hrs post - transfection GFP - DYRK3 - dN cells were either left untreated or exposed to GA ( 5 µM ) for 4 hrs . In untreated cells ( control ) , GFP - DYRK3 - dN is diffusely distributed in the cytosol and in the nucleus . Upon GA treatment , GFP - DYRK3 - dN forms perinuclear ( PN ) aggregates . Representative confocal images are shown . Scale bar is 10 µm .

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(F) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3-dN. 24 hrs post-transfection GFP-DYRK3-dN cells were either left untreated or exposed to GA (5 µM) for 4 hrs. In untreated cells (control), GFP-DYRK3-dN is diffusely distributed in the cytosol and in the nucleus. Upon GA treatment, GFP-DYRK3-dN forms perinuclear (PN) aggregates. Representative confocal images are shown. Scale bar is 10 µm.


( I ) mCherry - G3BP1 expressing HeLa cells were lipofected with cDNAs encoding for GFP - DYRK3 - dN or Sup35 - NM - GFP - DYRK3 - dN . 24 hrs post - transfection cells were treated for 45 min with sodium arsenite to induce SGs , followed by recovery for 4 hrs in presence of GA ( 5 µM ; rec . GA ) . Representative confocal microscopy images are shown . Nucleic acid was stained with DAPI . Scale bar is 10 µm .

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(I) mCherry-G3BP1 expressing HeLa cells were lipofected with cDNAs encoding for GFP-DYRK3-dN or Sup35-NM-GFP-DYRK3-dN. 24 hrs post-transfection cells were treated for 45 min with sodium arsenite to induce SGs, followed by recovery for 4 hrs in presence of GA (5 µM; rec. GA). Representative confocal microscopy images are shown. Nucleic acid was stained with DAPI. Scale bar is 10 µm.


( C ) Quantitation of the fluorescence intensity recovery after bleach of GFP - DYRK3 in G3BP1 - mCherry HeLa - Kyoto cells 24 h after transfection . Cells were treated with sodium arsenite ( 50 µM ) for 45 min to induce SGs , which were visualized with G3BP1 - mCherry ; then , sodium arsenite was removed and the cells were incubated in drug - free medium or in presence of GA ( 5 µM ) or of GSK ( 5 µM ) . FRAP was performed during the stress recovery period . A representative image of cells treated with arsenite followed by recovery in drug - free medium is shown . Arrowheads and dotted circle indicate the ROI . Scale bar is 5 µm . The mean of 13 FRAP curves ( recovery control ) , 12 FRAP curves ( recovery GA ) and 14 FRAP curves ( recovery GSK ) is shown in red ; the sem is shown in gray .

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(C) Quantitation of the fluorescence intensity recovery after bleach of GFP-DYRK3 in G3BP1-mCherry HeLa-Kyoto cells 24 h after transfection. Cells were treated with sodium arsenite (50 µM) for 45 min to induce SGs, which were visualized with G3BP1-mCherry; then, sodium arsenite was removed and the cells were incubated in drug-free medium or in presence of GA (5 µM) or of GSK (5 µM). FRAP was performed during the stress recovery period. A representative image of cells treated with arsenite followed by recovery in drug-free medium is shown. Arrowheads and dotted circle indicate the ROI. Scale bar is 5 µm. The mean of 13 FRAP curves (recovery control), 12 FRAP curves (recovery GA) and 14 FRAP curves (recovery GSK) is shown in red; the sem is shown in gray.


( B ) Confocal microscopy images of P525L FUS eGFP MNs treated with sodium arsenite for 2 hrs and showing colocalization of FUS with TIAR - positive stress granules . Scale bar is 10 µm . ( C ) P525L FUS eGFP MNs were treated with sodium arsenite for 2 hrs , followed by recovery in drug - free medium ( + rec . control ) or in presence of 17AAG ( 30 μM ; + rec . 17AAG ) or of GSK ( 5 μM ; + rec . GSK ) for 6 hrs . Quantitation of the number of SGs per cell in P525L FUS eGFP MNs . Number of P525L cells counted and n / condition : 2488 , n = 13 ( arsenite ) ; 1045 , n = 12 ( arsenite + rec . control ) ; 646 , n = 6 ( arsenite + rec . 17AAG ) ; 924 , n = 3 ( arsenite + rec . GSK ) , ± sem ( One - way ANOVA ) .

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(B) Confocal microscopy images of P525L FUS eGFP MNs treated with sodium arsenite for 2 hrs and showing colocalization of FUS with TIAR-positive stress granules. Scale bar is 10 µm. (C) P525L FUS eGFP MNs were treated with sodium arsenite for 2 hrs, followed by recovery in drug-free medium (+ rec. control) or in presence of 17AAG (30 μM; + rec. 17AAG) or of GSK (5 μM; +rec. GSK) for 6 hrs. Quantitation of the number of SGs per cell in P525L FUS eGFP MNs. Number of P525L cells counted and n/condition: 2488, n = 13 (arsenite); 1045, n = 12 (arsenite + rec. control); 646, n = 6 (arsenite + rec. 17AAG); 924, n = 3 (arsenite + rec. GSK), ± sem (One-way ANOVA).