A . Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus . Scale bar : 30 µm . B , C . ERK71 mutant fat bodies display increased nuclear area ( B , N > 160 cells , obtained from 5 independent fat bodies ) and increased nucleolar / nuclear area ratio ( C , N > 80 cells for nucleolus / nucleus ratio , obtained from 5 independent fat bodies ) , when compared to control fat bodies .

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

A. Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus. Scale bar: 30 µm. B, C. ERK71 mutant fat bodies display increased nuclear area (B, N>160 cells, obtained from 5 independent fat bodies) and increased nucleolar/nuclear area ratio (C, N>80 cells for nucleolus/nucleus ratio, obtained from 5 independent fat bodies), when compared to control fat bodies.


D - F . ERK7 expressing fat body clones ( D , marked by GFP , scale bar : 20 µm ) display reduced nuclear area ( E ) and reduced nucleolar / nuclear area ratio ( F ) , when compared to control cells . Nuclei were visualized by DAPI staining , nucleoli were visualized by anti - Fibrillarin antibodies ( N = 10 ) .

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

D-F. ERK7 expressing fat body clones (D, marked by GFP, scale bar: 20 µm) display reduced nuclear area (E) and reduced nucleolar/nuclear area ratio (F), when compared to control cells. Nuclei were visualized by DAPI staining, nucleoli were visualized by anti-Fibrillarin antibodies (N=10).


G , H . Expression analysis of Pol I ( G ) and Pol III ( H ) target RNAs by qRT - PCR . Expression of CDK7 was used for normalization ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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

G, H. Expression analysis of Pol I (G) and Pol III (H) target RNAs by qRT-PCR. Expression of CDK7 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).


A . ERK7 overexpression leads to phosphorylation of PWP1 , as shown by appearance of an extra band on Phos - tag Western blot ( marked with red arrow ) . Insulin - inducible bands ( dark blue ) remain unresponsive to ERK7 .

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

A. ERK7 overexpression leads to phosphorylation of PWP1, as shown by appearance of an extra band on Phos-tag Western blot (marked with red arrow). Insulin-inducible bands (dark blue) remain unresponsive to ERK7.


B . Representative immunofluorescent images of PWP1 localization in fat bodies of ERK71 mutant and control third instar larvae . Scale bar : 20 µm . C . PWP1 localization displays increased nucleolar / nuclear ratio in ERK71 mutant fat bodies compared to control ( N = 20 cells from 5 independent fat bodies per genotype ) .

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

B. Representative immunofluorescent images of PWP1 localization in fat bodies of ERK71 mutant and control third instar larvae. Scale bar: 20 µm. C. PWP1 localization displays increased nucleolar/nuclear ratio in ERK71 mutant fat bodies compared to control (N=20 cells from 5 independent fat bodies per genotype).


D . Representative immunofluorescent images of PWP1 localization in fat bodies of third instar larvae . ERK7 overexpression ( GFP marked clones ) alters the subcellular localization of PWP1 . Scale bar : 30 µm . E . PWP1 localization displays reduced nuclear / cytoplasmic ratio in ERK7 overexpressing clones , compared to control cells ( N = 10 cells from at least 8 different fat bodies per genotype ) .

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

D. Representative immunofluorescent images of PWP1 localization in fat bodies of third instar larvae. ERK7 overexpression (GFP marked clones) alters the subcellular localization of PWP1. Scale bar: 30 µm. E. PWP1 localization displays reduced nuclear/cytoplasmic ratio in ERK7 overexpressing clones, compared to control cells (N=10 cells from at least 8 different fat bodies per genotype).


F . Fat body specific depletion of PWP1 by RNAi leads to decreased TAG levels ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) .

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

F. Fat body specific depletion of PWP1 by RNAi leads to decreased TAG levels (N=4 replicates of ≥10 larvae/replicate for each genotype).


G . Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body ( CG - GAL4 ) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus , respectively . Scale bar : 50 µm . H , I . Fat body specific knockdown of PWP1 suppresses increased lipid droplet number ( H ; N > 30 cells per genotype ) and increased nuclear area ( I ; N > 45 cells per genotype ) caused by ERK7 knockdown ( BDSC 56939 ) .

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

G. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body (CG-GAL4) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. H, I. Fat body specific knockdown of PWP1 suppresses increased lipid droplet number (H; N>30 cells per genotype) and increased nuclear area (I; N>45 cells per genotype) caused by ERK7 knockdown (BDSC 56939).


A , B . Expression of sugarbabe is downregulated in fat bodies upon knockdown of PWP1 ( A ) and ectopic expression of ERK7 ( B ) ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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

A, B. Expression of sugarbabe is downregulated in fat bodies upon knockdown of PWP1 (A) and ectopic expression of ERK7 (B) (N=3 replicates of 10 fat bodies/replicate for each genotype).


C . ERK71 mutants fail to maximally downregulate sugarbabe expression upon fasting ( N = 4 replicates of 10 larvae / replicate for each genotype ) .

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

C. ERK71 mutants fail to maximally downregulate sugarbabe expression upon fasting (N=4 replicates of 10 larvae/replicate for each genotype).


D . Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK71 and sug mutants ( sug17Δ / Df ( 2R ) Exel7123 ) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus , respectively . Scale bar : 50 µm . E . sug mutant ( sug17Δ / Df ( 2R ) Exel7123 ) suppresses increased lipid droplet number observed in ERK71 mutants ( N > 20 ) .

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

D. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK71 and sug mutants (sug17Δ/Df(2R)Exel7123) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. E. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased lipid droplet number observed in ERK71 mutants (N>20).


F . Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG > ERK7 larvae ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) .

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

F. Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG>ERK7 larvae (N=4 replicates of ≥10 larvae/replicate for each genotype).


G . sug mutant ( sug17Δ / Df ( 2R ) Exel7123 ) suppresses increased nuclear size phenotype of ERK71 mutant fat body cells ( N ≥ 25 cells per genotype ) .

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

G. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased nuclear size phenotype of ERK71 mutant fat body cells (N ≥25 cells per genotype).


Ectopic Sugarbabe expression in the fat body partially restores the pupariation rate ( H ) ( p < 0 . 001 for the rescue , analyzed by Log - rank test , N = 4 replicates of 30 larvae / replicate for each genotype ) of CG > ERK7

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

Ectopic Sugarbabe expression in the fat body partially restores the pupariation rate (H) (p<0.001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype) of CG>ERK7 larvae


I . Ectopic Sugarbabe expression in the fat body partially restores the pupal volume of CG > ERK7 larvae ( I ) ( N = 4 replicates of 10 pupae / replicate for each genotype ) .

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

I. Ectopic Sugarbabe expression in the fat body partially restores the pupal volume of CG>ERK7 larvae (I) (N=4 replicates of 10 pupae/replicate for each genotype).


J . Addition of 20 % sucrose improves the growth rate and survival of CG > ERK7 larvae . Pupariation kinetics of CG > ERK7 larvae on 20 % yeast or 20 % yeast + 20 % sucrose diet ( p < 0 . 0001 for the rescue , analyzed by Log - rank test , N = 4 replicates of 30 larvae / replicate for each genotype and diet ) .

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

J. Addition of 20% sucrose improves the growth rate and survival of CG>ERK7 larvae. Pupariation kinetics of CG>ERK7 larvae on 20% yeast or 20% yeast + 20% sucrose diet (p<0.0001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype and diet).


F . Microscopic images of the Q97 - GFP fluorescence . Bars , 5 µm . G . The percentage of cells containing or lacking detectable aggregates was quantified . Cell boundaries are indicated by dashed lines .

O O O O O O B-SMALL_MOLECULE 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. Microscopic images of the Q97-GFP fluorescence. Bars, 5 µm. G. The percentage of cells containing or lacking detectable aggregates was quantified. Cell boundaries are indicated by dashed lines.


E . Q97 - GFP was expressed in mia40 - 3 and corresponding wild type cells for the times indicated . Cells were harvested , washed and survivors were counted after plating on glucose plates . Mean values and standard deviations of three replicates are shown .

O O B-SMALL_MOLECULE 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 O B-SMALL_MOLECULE O O O O O O O O O O O O O

E. Q97-GFP was expressed in mia40-3 and corresponding wild type cells for the times indicated. Cells were harvested, washed and survivors were counted after plating on glucose plates. Mean values and standard deviations of three replicates are shown.


B . Cell extracts were analyzed by Western blotting after shifting cultures to galactose for 16 h . 25 , Q25 - GFP ; 97 , Q97 - GFP ; ev , empty vector .

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

B. Cell extracts were analyzed by Western blotting after shifting cultures to galactose for 16 h. 25, Q25-GFP; 97, Q97-GFP; ev, empty vector.


C . Microscopy images of the indicated strains 12 h after shifting them to galactose . Note that in GAL - Mia40 cells the form and number of aggregates is very different to WT cells . Bar , 5 µm .

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

C. Microscopy images of the indicated strains 12 h after shifting them to galactose. Note that in GAL-Mia40 cells the form and number of aggregates is very different to WT cells. Bar, 5 µm.


E . PolyQ proteins were expressed for 24 h before survivors were counted . Mean values and standard deviations from three independent experiments are shown .

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

E. PolyQ proteins were expressed for 24 h before survivors were counted. Mean values and standard deviations from three independent experiments are shown.


B . Still frames after different times of growth indicate a uniform fluorescence in Q25 - GFP - expressing cells . Q97 - GFP expression in wild type leads to many small and scattered aggregates per cell . In GAL - Mia40 cells , Q97 - GFP accumulates to much larger intensities but then suddenly collapses into one single aggregate per cell . The arrow depicts aggregate formation . Bars , 4 µm . C . Quantified signal intensities in cells of the indicated mutants . In top and middle graphs , each curve corresponds to a single cell . Averaged signals ( mean + SEM ) in bottom graph ( N = 20 for WT , N = 17 cells for GAL - Mia40 ) .

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

B. Still frames after different times of growth indicate a uniform fluorescence in Q25-GFP-expressing cells. Q97-GFP expression in wild type leads to many small and scattered aggregates per cell. In GAL-Mia40 cells, Q97-GFP accumulates to much larger intensities but then suddenly collapses into one single aggregate per cell. The arrow depicts aggregate formation. Bars, 4 µm. C. Quantified signal intensities in cells of the indicated mutants. In top and middle graphs, each curve corresponds to a single cell. Averaged signals (mean + SEM) in bottom graph (N=20 for WT, N=17 cells for GAL-Mia40).


B . Q97 - GFP was expressed for 4 . 5 h in wild type and GAL - Mia40 cells in two biological replicates each . Cells were lysed with NP - 40 before soluble proteins were quantified . Mitochondrial proteins ( mito . ) and members of the mitochondrial carrier family ( carrier ) are visualized in a volcano plot . P - values were derived from a moderated t - test . Mitochondrial ( mito . ) and carrier proteins are indicated by yellow and red dots , respectively . Rnq1 is indicated in blue .

O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD 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 O B-SUBCELLULAR O O B-SUBCELLULAR 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 B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O O O O

B. Q97-GFP was expressed for 4.5 h in wild type and GAL-Mia40 cells in two biological replicates each. Cells were lysed with NP-40 before soluble proteins were quantified. Mitochondrial proteins (mito.) and members of the mitochondrial carrier family (carrier) are visualized in a volcano plot. P-values were derived from a moderated t-test. Mitochondrial (mito.) and carrier proteins are indicated by yellow and red dots, respectively. Rnq1 is indicated in blue.


I . Cells of the newly made strains were grown on galactose for 1 or 7 days , respectively . Cells were lysed before the distribution of Rnq1 in the soluble and pellet fractions was quantified . Shown are mean values and standard deviations of three independent replicates .

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

I. Cells of the newly made strains were grown on galactose for 1 or 7 days, respectively. Cells were lysed before the distribution of Rnq1 in the soluble and pellet fractions was quantified. Shown are mean values and standard deviations of three independent replicates.


C . ∆ ura3 cells expressing the Oxa1 - Ura3 reporter for the cytosolic accumulation of mitochondrial precursor proteins ( Hansen et al . , 2018 ) were transformed with plasmids expressing the proteins TDP - 43 , the TDP - 43Q331K mutant , Q25 - GFP , Q97 - GFP , α - synuclein , FUS or GFP from a constitutive glyceraldehyde - 3 - phosphate dehydrogenase ( GPD ) promoter . Note that the expression of all aggregation - prone proteins ( indicated by red arrows ) allows cells to grow on uracil - deficient plates indicating the cytosolic accumulation of the Oxa1 - Ura3 fusion protein .

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

C. ∆ura3 cells expressing the Oxa1-Ura3 reporter for the cytosolic accumulation of mitochondrial precursor proteins (Hansen et al., 2018) were transformed with plasmids expressing the proteins TDP-43, the TDP-43Q331K mutant, Q25-GFP, Q97-GFP, α-synuclein, FUS or GFP from a constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. Note that the expression of all aggregation-prone proteins (indicated by red arrows) allows cells to grow on uracil-deficient plates indicating the cytosolic accumulation of the Oxa1-Ura3 fusion protein.


E . Wild type cells were transformed with plasmids for the expression of the indicated proteins . Q97 - GFPnF is a non - fluorescent version of Q97 - GFP . Close proximity of the two split GFP parts results in fluorescence which was visualized by microscopy . Shown are mean values and standard deviations of three replicates . Bar , 5 µm .

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

E. Wild type cells were transformed with plasmids for the expression of the indicated proteins. Q97-GFPnF is a non-fluorescent version of Q97-GFP. Close proximity of the two split GFP parts results in fluorescence which was visualized by microscopy. Shown are mean values and standard deviations of three replicates. Bar, 5 µm.


B . The open reading frames of the indicated proteins were cloned downstream of a GAL promoter in plasmids and transformed into cells harboring the Q97 - GFP expression plasmid . Cells harboring an empty vector ( ev ) were used for control . Cells were shifted to galactose medium overnight before they were dropped onto glucose or galactose plates . If cells are directly dropped from lactate medium onto galactose plates , only individual cells ( indicated by red protein names ) escaped the polyQ toxicity ( see Fig . EV4C ) .

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

B. The open reading frames of the indicated proteins were cloned downstream of a GAL promoter in plasmids and transformed into cells harboring the Q97-GFP expression plasmid. Cells harboring an empty vector (ev) were used for control. Cells were shifted to galactose medium overnight before they were dropped onto glucose or galactose plates. If cells are directly dropped from lactate medium onto galactose plates, only individual cells (indicated by red protein names) escaped the polyQ toxicity (see Fig. EV4C).


D . Cells expressing the indicated carrier proteins from GAL promoters were dropped onto galactose plates . Cell growth was quantified by densitometry from one representative plate . OM refers to carrier proteins that were expressed as fusions with an N - terminal outer membrane anchor . See Fig . EV4D , E for scans of the respective plates .

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

D. Cells expressing the indicated carrier proteins from GAL promoters were dropped onto galactose plates. Cell growth was quantified by densitometry from one representative plate. OM refers to carrier proteins that were expressed as fusions with an N-terminal outer membrane anchor. See Fig. EV4D, E for scans of the respective plates.


B . SH - SY5Y cells co - expressing Q97 - GFP with MIA40 ( middle row ) , ∆ N - MIA40 ( lower row ) or a mock - transfected control ( upper row ) were analyzed by immunocytochemistry and super - resolution microscopy . The magnification shows an enlarged section of the TOM20 staining . TOM20 and TRAP1 serve as markers for the mitochondrial outer membrane and the matrix , respectively . Bars , 20 µm . Magnifications : 40fold of a 63x objective field .

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

B. SH-SY5Y cells co-expressing Q97-GFP with MIA40 (middle row), ∆N-MIA40 (lower row) or a mock-transfected control (upper row) were analyzed by immunocytochemistry and super-resolution microscopy. The magnification shows an enlarged section of the TOM20 staining. TOM20 and TRAP1 serve as markers for the mitochondrial outer membrane and the matrix, respectively. Bars, 20 µm. Magnifications: 40fold of a 63x objective field.


D . SH - SY5Y cells co - expressing Q97 - GFP with MIA40 ( upper row ) or ∆ N - MIA40 ( lower row ) were analyzed by immunocytochemistry and super - resolution microscopy . The magnification shows an enlarged section of the TRAP1 staining used as a marker for the mitochondrial matrix , respectively . Bars , 20 µm . Magnifications : 40fold of a 63x objective field .

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

D. SH-SY5Y cells co-expressing Q97-GFP with MIA40 (upper row) or ∆N-MIA40 (lower row) were analyzed by immunocytochemistry and super-resolution microscopy. The magnification shows an enlarged section of the TRAP1 staining used as a marker for the mitochondrial matrix, respectively. Bars, 20 µm. Magnifications: 40fold of a 63x objective field.


( A ) Mice bearing established A375 were treated with vehicle ( n = 7 ) , PLX4720 ( n = 5 ) bevacizumab ( n = 7 ) or COMBO ( n = 5 ) . Tumor growth is expressed as % change of the initial tumor , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( PLX4720 P = 0 . 0049 ; bevacizumab P = 0 . 0003 ; P = 4 . 86E - 10 ) ŦP < 0 . 05 compared to PLX4720 ( P = 0 . 015 ) . ( B ) Mice bearing established COLO205 were treated with vehicle ( n = 7 ) , PLX4720 ( n = 8 ) bevacizumab ( n = 7 ) or COMBO ( n = 8 ) . Tumor growth is expressed as % change of the initial tumor , * * * P < 0 . 001 versus vehicle ( PLX4720 P = 2 . 75E - 05 ; bevacizumab P = 2 . 96E - 05 ; COMBO P = 2 . 13E - 07 ) Ŧp < 0 . 05 compared to PLX4720 ( P = 0 . 039 ) .

O O O B-ORGANISM O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE 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 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 B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE O O O O O O O O

(A) Mice bearing established A375 were treated with vehicle (n=7), PLX4720 (n=5) bevacizumab (n=7) or COMBO (n=5). Tumor growth is expressed as % change of the initial tumor, **P < 0.01, ***P < 0.001 versus vehicle (PLX4720 P = 0.0049; bevacizumab P = 0.0003; P = 4.86E-10) ŦP < 0.05 compared to PLX4720 (P = 0.015).(B) Mice bearing established COLO205 were treated with vehicle (n=7), PLX4720 (n=8) bevacizumab (n=7) or COMBO (n=8). Tumor growth is expressed as % change of the initial tumor, ***P < 0.001 versus vehicle (PLX4720 P = 2.75E-05; bevacizumab P = 2.96E-05; COMBO P = 2.13E-07) Ŧp < 0.05 compared to PLX4720 (P = 0.039).


( C ) Melanoma MC - 1 cells ( 5 x 105 ) were injected into the tail vein of CD1 mice . Lung colonization was assayed by HE staining and calculating the number of nodules and their total area normalized per the total area of the lungs . The mice analyzed were : start point , n = 6 ; vehicle , n = 7 ; PLX4720 , n = 6 ; bevacizumab , n = 6 ; COMBO , n = 7 , * P < 0 . 001 versus vehicle ( P = 0 . 031 ) .

O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-ORGANISM O B-TISSUE 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 B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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

(C) Melanoma MC-1 cells (5 x 105) were injected into the tail vein of CD1 mice. Lung colonization was assayed by HE staining and calculating the number of nodules and their total area normalized per the total area of the lungs. The mice analyzed were: start point, n=6; vehicle, n=7; PLX4720, n=6; bevacizumab, n=6; COMBO, n=7, *P < 0.001 versus vehicle (P = 0.031).


( D ) Representative images of tumor cell proliferation determined by immunofluorescence Ki67 staining in A375 xenografts treated as indicated . Bar graphs indicate the Ki67 + area / tumor area ( n = 4 tumors ) , * * * P < 0 . 001 versus vehicle ( PLX4720 P = 2 . 83E - 08 ; bevacizumab P = 4 . 25E - 13 ; COMBO P = 6 . 16E - 08 ) . ( E ) Representative images of tumor cell apoptosis determined by immunofluorescence staining with TUNEL in A375 xenografts treated as indicated . Bar graphs indicate the TUNEL + area / tumor area ( n = 3 tumors ) . * * * P < 0 . 001 versus vehicle ( P = 6 . 2 . 7E - 10 ) .

O O O O O O B-TISSUE O B-EXP_ASSAY O O O B-GENEPROD O O B-CELL_LINE B-ORGANISM O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O B-SMALL_MOLECULE 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 B-TISSUE O O O O O O O B-EXP_ASSAY O B-CELL_LINE B-ORGANISM O O O O O O O O B-EXP_ASSAY O O O B-TISSUE O O O O O B-TISSUE 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) Representative images of tumor cell proliferation determined by immunofluorescence Ki67 staining in A375 xenografts treated as indicated. Bar graphs indicate the Ki67+ area/tumor area (n=4 tumors), ***P < 0.001 versus vehicle (PLX4720 P = 2.83E-08; bevacizumab P = 4.25E-13; COMBO P = 6.16E-08).(E) Representative images of tumor cell apoptosis determined by immunofluorescence staining with TUNEL in A375 xenografts treated as indicated. Bar graphs indicate the TUNEL+ area/tumor area (n=3 tumors). ***P < 0.001 versus vehicle (P = 6.2.7E-10).


( A ) Representative images of vasculature stained by an antibody anti - CD31 in A375 xenografts treated as indicated . Bar graphs indicate quantitative micro vessel density ( MVD ) and micro vessel area ( MVA ) analysis ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( MVD : bevacizumab P = 5 . 94E - 25 ; COMBO P = 1 . 84E - 04 ) ( MVA : PLX4720 P = 4 . 98E - 09 ; bevacizumab P = 1 . 16E - 19 ; COMBO P = 3 . 00E - 08 ) .

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

(A) Representative images of vasculature stained by an antibody anti-CD31 in A375 xenografts treated as indicated. Bar graphs indicate quantitative micro vessel density (MVD) and micro vessel area (MVA) analysis (n=5 tumors), ***P < 0.001 versus vehicle (MVD: bevacizumab P = 5.94E-25; COMBO P = 1.84E-04) (MVA: PLX4720 P = 4.98E-09; bevacizumab P = 1.16E-19; COMBO P = 3.00E-08).


( B ) Representative images of vessel lumen in A375 xenografts treated as indicated . Bar graphs indicate the quantitative analysis of lumen diameters in A375 and COLO205 xenografts ( n = 3 tumors ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( A375 : PLX4720 P = 0 . 0078 ; COMBO P = 0 . 0012 ) ( COLO205 : PLX4720 P = 2 . 01E - 06 ; COMBO P = 1 . 33E - 07 ) .

O O O O O O B-TISSUE I-TISSUE O B-CELL_LINE B-ORGANISM O O O O O O O O O O O O O O B-CELL_LINE O B-CELL_LINE B-ORGANISM O O O O B-TISSUE O O O O 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-CELL_LINE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

(B) Representative images of vessel lumen in A375 xenografts treated as indicated. Bar graphs indicate the quantitative analysis of lumen diameters in A375 and COLO205 xenografts (n=3 tumors), *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (A375: PLX4720 P = 0.0078; COMBO P = 0.0012) (COLO205: PLX4720 P = 2.01E-06; COMBO P = 1.33E-07).


( C ) Representative images of perfusing fluorescent beads and their relationship with microvessels in A375 xenografts treated as indicated . Bar graphs indicate the % of CD31 + vessels co - stained with fluorescent beads ( n = 4 tumors ) , * * P < 0 . 01 versus vehicle ( PLX4720 P = 0 . 0031 ; COMBO P = 0 . 0033 ) .

O O O O O O O O O O O O O B-TISSUE O B-CELL_LINE B-ORGANISM 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-TISSUE 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

(C) Representative images of perfusing fluorescent beads and their relationship with microvessels in A375 xenografts treated as indicated. Bar graphs indicate the % of CD31+ vessels co-stained with fluorescent beads (n=4 tumors), **P < 0.01 versus vehicle (PLX4720 P = 0.0031; COMBO P = 0.0033).


( D ) Representative images of hypoxia marker pimonidazole in A375 xenografts treated as indicated . Bar graphs show the % of tumor hypoxic area in A375 and COLO205 xenografts ( n = 5 tumors ) , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( A375 : PLX4720 P = 6 . 94E - 12 ; COMBO P = 3 . 34E - 12 ) ( COLO205 : PLX4720 P = 0 . 0015 ; bevacizumab P = 0 . 0004 ; COMBO P = 0 . 0003 ) .

O O O O O O O O B-EXP_ASSAY O B-CELL_LINE B-ORGANISM O O O O O O O O O O B-TISSUE O O O B-CELL_LINE O B-CELL_LINE B-ORGANISM O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(D) Representative images of hypoxia marker pimonidazole in A375 xenografts treated as indicated. Bar graphs show the % of tumor hypoxic area in A375 and COLO205 xenografts (n=5 tumors), **P < 0.01, ***P < 0.001 versus vehicle (A375: PLX4720 P = 6.94E-12; COMBO P = 3.34E-12) (COLO205: PLX4720 P = 0.0015; bevacizumab P = 0.0004; COMBO P = 0.0003).


( D ) GSEA enrichment plots for " Reactome Cytokine Signaling in Immune System " and " Reactome Innate Immune System " ( upper panels ) and " Reactome Extracellular Matrix Organization " and " Reactome Collagen Formation " ( lower panels ) highlight significant enrichment of the pathways relative to the immune response and a decreased expression of the pathways relative to extracellular matrix remodeling in COMBO treated tumors as compared to the other treatments ( vehicle , PLX4720 , bevacizumab ) .

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-TISSUE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O

(D) GSEA enrichment plots for "Reactome Cytokine Signaling in Immune System" and "Reactome Innate Immune System" (upper panels) and "Reactome Extracellular Matrix Organization" and "Reactome Collagen Formation" (lower panels) highlight significant enrichment of the pathways relative to the immune response and a decreased expression of the pathways relative to extracellular matrix remodeling in COMBO treated tumors as compared to the other treatments (vehicle, PLX4720, bevacizumab).


( E ) Heatmap representation of gene expression changes within the " Reactome Innate Immune System " ( left panel ) and " Reactome Extracellular matrix Organization " ( right panel ) gene set . Genes in heatmaps are shown in rows and samples are shown in columns . Expression level is represented as a gradient from high ( red ) to low ( blue ) . V , P , B and C respectively indicate vehicle , PLX4720 , bevacizumab and COMBO .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE O O O

(E) Heatmap representation of gene expression changes within the "Reactome Innate Immune System" (left panel) and "Reactome Extracellular matrix Organization" (right panel) gene set. Genes in heatmaps are shown in rows and samples are shown in columns. Expression level is represented as a gradient from high (red) to low (blue). V, P, B and C respectively indicate vehicle, PLX4720, bevacizumab and COMBO.


( A ) Representative images of leukocytes infiltration determined by immunofluorescence CD45 staining in of A375 xenografts treated as indicated . Bar graphs indicate the CD45 + area / tumor area in A375 and COLO205 xenografts ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( A375 : P = 2 . 19E - 20 ) ( COLO205 : P = 1 . 81E - 06 ) . ( B ) Representative images of macrophage infiltration determined by F4 / 80 immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the F4 / 80 + area / tumor area in A375 and COLO205 xenografts ( n = 5 tumors ) * * * P < 0 . 001 versus vehicle ( A375 : COMBO P = 3 . 42E - 13 ) ( COLO205 : COMBO P = 1 . 94E - 06 ) .

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

(A) Representative images of leukocytes infiltration determined by immunofluorescence CD45 staining in of A375 xenografts treated as indicated. Bar graphs indicate the CD45+ area/tumor area in A375 and COLO205 xenografts (n=5 tumors), ***P < 0.001 versus vehicle (A375: P = 2.19E-20) (COLO205: P = 1.81E-06).(B) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the F4/80+ area/tumor area in A375 and COLO205 xenografts (n=5 tumors) ***P < 0.001 versus vehicle (A375: COMBO P = 3.42E-13) (COLO205: COMBO P = 1.94E-06).


( C ) A375 xenograft tumors after 14 days of treatment were subjected to FACS and tumor infiltrate was analyzed . Gating strategy for CD45 + cells and graph showing the quantification of FACS analysis of infiltrating CD45 + leukocytes in vehicle ( n = 4 tumors ) compared to COMBO ( n = 6 tumors ) treated tumors , * * P < 0 . 01 versus vehicle ( P = 0 . 0064 ) .

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

(C) A375 xenograft tumors after 14 days of treatment were subjected to FACS and tumor infiltrate was analyzed. Gating strategy for CD45+ cells and graph showing the quantification of FACS analysis of infiltrating CD45+ leukocytes in vehicle (n=4 tumors) compared to COMBO (n=6 tumors) treated tumors, **P < 0.01 versus vehicle (P = 0.0064).


( D ) Graph showing the quantification of FACS analysis of infiltrating F4 / 80 + macrophages and CD11c + MHCII + dendritic cells in vehicle ( n = 4 tumors ) compared to COMBO ( n = 6 tumors ) treated tumors , * * P < 0 . 01 versus vehicle ( TAM P = 0 . 0061 ; DC P = 0 . 0091 ) .

O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O B-TISSUE O O 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) Graph showing the quantification of FACS analysis of infiltrating F4/80+ macrophages and CD11c+MHCII+ dendritic cells in vehicle (n=4 tumors) compared to COMBO (n=6 tumors) treated tumors, **P < 0.01 versus vehicle (TAM P = 0.0061; DC P = 0.0091).


( A ) Real - time quantitative PCR of the indicated genes ( M1 - like and M2 - like macrophages markers ) of A375 xenograft treated with PLX4720 , bevacizumab or COMBO . Data are presented as expression fold change ( log2 ) compared with vehicle after normalization for housekeeping gene TBP ( n = 3 tumors ) , ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( PLX4720 : m - Arg1 P = 5 . 65E - 06 ) ( bevacizumab : m - Ccl5 P = 0 . 025 ) ( COMBO : m - Ccl5 P = 0 . 0070 ; m - Cd40 P = 0 . 00099 ; m - Cxcl10 P = 0 . 043 ; m - Cxcl9 P = 0 . 046 ; m - Il1b P = 0 . 0367 ; m - Stat1 P = 0 . 049 ; m - Tlr2 = 0 . 00182 ; m - Arg - 1 P = 0 . 0013 ) .

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

(A) Real-time quantitative PCR of the indicated genes (M1-like and M2-like macrophages markers) of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) compared with vehicle after normalization for housekeeping gene TBP (n=3 tumors), (*P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (PLX4720: m-Arg1 P = 5.65E-06) (bevacizumab: m-Ccl5 P = 0.025) (COMBO: m-Ccl5 P = 0.0070; m-Cd40 P = 0.00099; m-Cxcl10 P = 0.043; m-Cxcl9 P = 0.046; m-Il1b P = 0.0367; m-Stat1 P = 0.049; m-Tlr2 = 0.00182; m-Arg-1 P = 0.0013).


( B ) Representative images of COMBO - treated tumor sections co - stained with an antibody against CD68 and an antibody anti - CCR7 ( M1 - like polarization ) or anti - CD206 ( M2 - like polarization ) .

O O O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O

(B) Representative images of COMBO-treated tumor sections co-stained with an antibody against CD68 and an antibody anti-CCR7 (M1-like polarization) or anti-CD206 (M2-like polarization).


( C ) Graphs showing the quantification of FACS analysis of infiltrating F4 / 80 + tumor macrophages and average percentage of median fluorescence intensity ( MFI ) of TNFα + or INOS + on F4 / 80 + cells after ex vivo stimulation with PMA and ionomycin in vehicle ( n = 4 tumors ) and COMBO ( n = 6 tumors ) , * P < 0 . 05 versus vehicle ( TNFα P = 0 . 037 ; INOS = 0 . 038 ) .

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

(C) Graphs showing the quantification of FACS analysis of infiltrating F4/80+ tumor macrophages and average percentage of median fluorescence intensity (MFI) of TNFα+ or INOS+ on F4/80+ cells after ex vivo stimulation with PMA and ionomycin in vehicle (n=4 tumors) and COMBO (n=6 tumors), *P < 0.05 versus vehicle (TNFα P = 0.037; INOS = 0.038).


( D ) Graphs showing the quantification of FACS analysis of infiltrating CD11b + Ly6C + tumor monocytes and average percentage of MFI of TNFα + or INOS + on CD11b + Ly6C + cells after ex vivo stimulation with PMA and ionomycin in vehicle ( n = 4 tumors ) and COMBO ( n = 6 tumors ) , * P < 0 . 05 versus vehicle ( TNFα P = 0 . 048 ; INOS = 0 . 038 ) .

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

(D) Graphs showing the quantification of FACS analysis of infiltrating CD11b+Ly6C+ tumor monocytes and average percentage of MFI of TNFα+ or INOS+ on CD11b+Ly6C+ cells after ex vivo stimulation with PMA and ionomycin in vehicle (n=4 tumors) and COMBO (n=6 tumors), *P < 0.05 versus vehicle (TNFα P = 0.048; INOS = 0.038).


( E ) Graph showing the quantification of circulating CD11b + Ly6C + monocytes in peripheral blood analyzed by FACS in vehicle - ( n = 4 tumors ) and COMBO - ( n = 6 tumors ) treated tumors .

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

(E) Graph showing the quantification of circulating CD11b+Ly6C+ monocytes in peripheral blood analyzed by FACS in vehicle- (n=4 tumors) and COMBO- (n=6 tumors) treated tumors.


( F ) Cell viability measured as fluorescence intensity of ZsGreen - A375 cells co - cultured with cells isolated from xenograft treated with vehicle or COMBO with after 48h . Data are representative of one experiment out of two done in triplicate * P < 0 . 05 versus vehicle ( P = 0 . 046 ) .

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

(F) Cell viability measured as fluorescence intensity of ZsGreen-A375 cells co-cultured with cells isolated from xenograft treated with vehicle or COMBO with after 48h. Data are representative of one experiment out of two done in triplicate *P < 0.05 versus vehicle (P = 0.046).


( A ) Representative images of collagen deposition determined by collagen I immunofluorescent staining in A375 xenografts treated as indicated . Bar graphs indicate the collagen I + area / tumor area ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 2 . 93E - 06 ) . ( B ) Representative images of CAF density determined by α - SMA immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate α - SMA + area / tumor area ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 0 . 0006 ) . ( C ) Representative images of Lysyl Oxidase enzyme determined by LOX immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the LOX + area / tumor area ( n = 3 tumors ) , * P < 0 . 05 versus vehicle ( bevacizumab P = 0 . 033 ; COMBO P = 0 . 040 ) .

O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-CELL_LINE B-ORGANISM O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O 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 I-GENEPROD I-GENEPROD O O O B-CELL_LINE B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O B-TISSUE O O O O O O O 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 B-CELL_LINE B-ORGANISM O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O B-TISSUE 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

(A) Representative images of collagen deposition determined by collagen I immunofluorescent staining in A375 xenografts treated as indicated. Bar graphs indicate the collagen I+ area/tumor area (n=5 tumors), ***P < 0.001 versus vehicle (P = 2.93E-06).(B) Representative images of CAF density determined by α-SMA immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate α-SMA+ area/tumor area (n=5 tumors), ***P < 0.001 versus vehicle (P = 0.0006).(C) Representative images of Lysyl Oxidase enzyme determined by LOX immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the LOX+ area/tumor area (n=3 tumors), *P < 0.05 versus vehicle (bevacizumab P = 0.033; COMBO P = 0.040).


( E ) Quantitative Real - time PCR of h - TGFB1 and m - Tgfb1 of A375 xenograft treated with PLX4720 , bevacizumab or COMBO . Data are presented as expression fold change ( log2 ) compared with vehicle after normalization for the housekeeping gene TBP ( n = 3 tumors ) , * P < 0 . 05 versus vehicle ( P = 0 . 043 ) .

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

(E) Quantitative Real-time PCR of h-TGFB1 and m-Tgfb1 of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) compared with vehicle after normalization for the housekeeping gene TBP (n=3 tumors), *P < 0.05 versus vehicle (P = 0.043).


( F ) Western blot for human TGFβ in total protein extract from A375 tumors treated as indicate . Vinculin was used as an internal control .

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

(F) Western blot for human TGFβ in total protein extract from A375 tumors treated as indicate. Vinculin was used as an internal control.


( A ) Effect of clodronate liposomes on tumor growth inhibition induced by COMBO treatment in in mice bearing A375 tumors . Tumor growth is expressed as % change of the initial tumor . Macrophage depletion by clodronate liposome enhanced the tumor growth of tumors treated with the combination of PLX4720 and bevacizumab ( n = 5 mice / group ) , * P < 0 . 05 , * * * P < 0 . 001 versus vehicle ( vehicle Clodronate P = 0 . 049 , COMBO P = 5 . 72E - 06 ) ŦP < 0 . 05 compared to COMBO ( P = 0 . 033 ) .

O O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O B-ORGANISM O B-CELL_LINE B-TISSUE O B-TISSUE O O O O O O O O O B-TISSUE O B-CELL_TYPE O O B-SMALL_MOLECULE O O O B-TISSUE O O B-TISSUE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 O O O O O O O O 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) Effect of clodronate liposomes on tumor growth inhibition induced by COMBO treatment in in mice bearing A375 tumors. Tumor growth is expressed as % change of the initial tumor. Macrophage depletion by clodronate liposome enhanced the tumor growth of tumors treated with the combination of PLX4720 and bevacizumab (n=5 mice/group), *P < 0.05, ***P < 0.001 versus vehicle (vehicle Clodronate P = 0.049, COMBO P = 5.72E-06) ŦP < 0.05 compared to COMBO (P = 0.033).


( B ) Representative images of macrophage infiltration determined by F4 / 80 immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the F4 / 80 + area / tumor ( n = 3 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 1 . 53E - 25 ) . ( C ) Representative images of collagen deposition determined by collagen I immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the collagen I + area / tumor ( n = 3 tumors ) .

O O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O B-TISSUE O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O B-CELL_LINE B-ORGANISM O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-TISSUE O O O O B-TISSUE O O

(B) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the F4/80+ area/tumor (n=3 tumors), ***P < 0.001 versus vehicle (P = 1.53E-25).(C) Representative images of collagen deposition determined by collagen I immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the collagen I+ area/tumor (n=3 tumors).


( D ) Waterfall plots showing the percent change in volume ( relative to the initial tumor volume ) for the individual A375 xenografts in each treatment group ( vehicle , PLX4720 , bevacizumab and COMBO ) from week 1 to week 6 .

O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-CELL_LINE B-ORGANISM O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O

(D) Waterfall plots showing the percent change in volume (relative to the initial tumor volume) for the individual A375 xenografts in each treatment group (vehicle, PLX4720, bevacizumab and COMBO) from week 1 to week 6.


( E ) Representative images of macrophage infiltration determined by F4 / 80 immunofluorescence staining in a responder and a relapsing A375 xenografts . Bar graphs indicate the F4 / 80 + area / tumor ( n = 3 mice ) , * * * P < 0 . 001 versus vehicle ( P = 1 . 25E - 06 ) . ( F ) Representative images of collagen deposition determined by collagen I immunofluorescence staining in a responder and a relapsing A375 xenografts . Bar graphs indicate the collagen I + area / tumor ( n = 3 mice ) , * * * P < 0 . 001 versus vehicle ( P = 4 . 03E - 05 ) .

O O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-CELL_LINE B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE 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 O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-CELL_LINE B-ORGANISM O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-TISSUE 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

(E) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in a responder and a relapsing A375 xenografts. Bar graphs indicate the F4/80+ area/tumor (n=3 mice), ***P < 0.001 versus vehicle (P = 1.25E-06).(F) Representative images of collagen deposition determined by collagen I immunofluorescence staining in a responder and a relapsing A375 xenografts. Bar graphs indicate the collagen I+ area/tumor (n=3 mice), ***P < 0.001 versus vehicle (P = 4.03E-05).


( G ) Real - time quantitative PCR of h - TGFB1 of A375 xenograft treated with PLX4720 , bevacizumab or COMBO . Data are presented as expression fold change ( log2 ) of relapsing tumors compared to responder tumors after normalization for housekeeping gene TBP ( n = 3 tumors ) * * * P < 0 . 001 versus vehicle ( P = 0 . 0006 ) .

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

(G) Real-time quantitative PCR of h-TGFB1 of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) of relapsing tumors compared to responder tumors after normalization for housekeeping gene TBP (n=3 tumors) ***P < 0.001 versus vehicle (P = 0.0006).


A . Sorbitol stimulates YAP phosphorylation . HEK293A cells were cultured in the presence or absence of serum , and treated with 0 . 2 M sorbitol for the indicated time points . Phos - tag gel was used to assess total YAP phosphorylation based on mobility shift . Note that YAP and TAZ are recognized by the same antibody . l . e . denotes long exposure of YAP and TAZ .

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

A. Sorbitol stimulates YAP phosphorylation. HEK293A cells were cultured in the presence or absence of serum, and treated with 0.2 M sorbitol for the indicated time points. Phos-tag gel was used to assess total YAP phosphorylation based on mobility shift. Note that YAP and TAZ are recognized by the same antibody. l.e. denotes long exposure of YAP and TAZ.


B . Osmotic stress induces LATS phosphorylation . HEK293A cells were treated with 0 . 2 M sorbitol for 30 or 60 minutes in the presence or absence of serum . LATS phosphorylation at the hydrophobic motif ( HM ) was detected with the phospho - specific pLATS antibody .

O O O O O O O O B-CELL_LINE 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 O

B. Osmotic stress induces LATS phosphorylation. HEK293A cells were treated with 0.2 M sorbitol for 30 or 60 minutes in the presence or absence of serum. LATS phosphorylation at the hydrophobic motif (HM) was detected with the phospho-specific pLATS antibody.


C . Osmotic stress increases LATS kinase activity . HEK293A cells were treated with 0 . 2 M sorbitol for 30 minutes . Lats1 was immunoprecipitated and an in vitro kinase assay was performed using recombinant GST - YAP as a substrate . Phosphorylation of YAP was determined by immunoblotting with phospho - YAP ( S127 ) antibody . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

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

C. Osmotic stress increases LATS kinase activity. HEK293A cells were treated with 0.2 M sorbitol for 30 minutes. Lats1 was immunoprecipitated and an in vitro kinase assay was performed using recombinant GST-YAP as a substrate. Phosphorylation of YAP was determined by immunoblotting with phospho-YAP (S127) antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


D . Osmotic stress does not lead to YAP cytoplasmic localization . HEK293A cells were treated with 0 . 2 M sorbitol for 1 hr in the presence of serum . YAP subcellular localization was determined by immunofluorescence staining ( red ) . DAPI ( blue ) was used to stain for DNA . Scale bar : 20 μm . Quantification of more nuclear ( N > C ) or more cytosolic ( N < C ) YAP signal is determined with randomly chosen fields , each with approximately 100 cells .

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

D. Osmotic stress does not lead to YAP cytoplasmic localization. HEK293A cells were treated with 0.2 M sorbitol for 1 hr in the presence of serum. YAP subcellular localization was determined by immunofluorescence staining (red). DAPI (blue) was used to stain for DNA. Scale bar: 20 μm. Quantification of more nuclear (N > C) or more cytosolic (N < C) YAP signal is determined with randomly chosen fields, each with approximately 100 cells.


E . Osmotic stress does not reduce YAP target gene expression . HEK293A cells were treated with 0 . 2 M sorbitol for 4 hr . mRNA levels of CTGF and CYR61 were measured by quantitative RT - PCR and normalized to GAPDH control . Data are presented as mean ± SEM . n . s means p > 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

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

E. Osmotic stress does not reduce YAP target gene expression. HEK293A cells were treated with 0.2 M sorbitol for 4 hr. mRNA levels of CTGF and CYR61 were measured by quantitative RT-PCR and normalized to GAPDH control. Data are presented as mean ± SEM. n.s means p > 0.05 (two tailed student's t-test, n = 3).


F . Osmotic stress does not disrupt the interaction between YAP and TEAD1 . HEK293A cells were treated with 0 . 2 M sorbitol for 1 hr . Endogenous YAP was immunoprecipitated with YAP antibody , and TEAD1 and YAP were detected by Western blot . Cells were serum starved for 1 hr as indicated ( - serum ) .

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

F. Osmotic stress does not disrupt the interaction between YAP and TEAD1. HEK293A cells were treated with 0.2 M sorbitol for 1 hr. Endogenous YAP was immunoprecipitated with YAP antibody, and TEAD1 and YAP were detected by Western blot. Cells were serum starved for 1 hr as indicated (- serum).


G . Osmotic stress does not increase the interaction between YAP and 14 - 3 - 3 . HEK293A cells were transiently transfected with Flag - YAP and Myc - 14 - 3 - 3 , then treated with 0 . 2 M sorbitol for the indicated time points . Myc - 14 - 3 - 3 was immunoprecipitated and the co - precipitated Flag - YAP was detected .

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

G. Osmotic stress does not increase the interaction between YAP and 14-3-3. HEK293A cells were transiently transfected with Flag-YAP and Myc-14-3-3, then treated with 0.2 M sorbitol for the indicated time points. Myc-14-3-3 was immunoprecipitated and the co-precipitated Flag-YAP was detected.


A . Osmotic stress induces YAP nuclear translocation . HEK293A cells were serum starved for 1 hr followed by 0 . 2 M sorbitol treatment for 1 hr . YAP / TAZ ( red , upper panels ) or YAP ( red , lower panels ) were stained with two specific antibodies . DAPI ( blue ) was used to visualize cell nuclei . Scale bar : 20 μm . Quantification of more nuclear ( N > C ) or more cytosolic ( N < C ) YAP signal is determined with randomly chosen fields .

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

A. Osmotic stress induces YAP nuclear translocation. HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for 1 hr. YAP/TAZ (red, upper panels) or YAP (red, lower panels) were stained with two specific antibodies. DAPI (blue) was used to visualize cell nuclei. Scale bar: 20 μm. Quantification of more nuclear (N > C) or more cytosolic (N < C) YAP signal is determined with randomly chosen fields.


B . Osmotic stress induces TAZ nuclear translocation . YAP knockout ( KO ) HEK293A cells were serum starved for 1 hr followed by 0 . 2 M sorbitol treatment for 1 hr . TAZ localization was determined by immunofluorescence staining with the YAP / TAZ antibody ( red ) . Scale bar : 20 μm .

O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

B. Osmotic stress induces TAZ nuclear translocation. YAP knockout (KO) HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for 1 hr. TAZ localization was determined by immunofluorescence staining with the YAP/TAZ antibody (red). Scale bar: 20 μm.


C . Osmotic stress induces a transient YAP nuclear translocation . HEK293A cells were serum starved for 1 hr followed by 0 . 2 M sorbitol treatment for the indicated time points .

O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O

C. Osmotic stress induces a transient YAP nuclear translocation. HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for the indicated time points.


D . Osmotic stress induces YAP target gene expression . Wild type ( WT ) or YAP / TAZ double knockout ( Y / T KO ) HEK293A cells were serum starved for 1 hr and treated with 0 . 2 M sorbitol for 4 hr . mRNA levels of CTGF and CYR61 were measured by quantitative RT - PCR and normalized to GAPDH control . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

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

D. Osmotic stress induces YAP target gene expression. Wild type (WT) or YAP/TAZ double knockout (Y/T KO) HEK293A cells were serum starved for 1 hr and treated with 0.2 M sorbitol for 4 hr. mRNA levels of CTGF and CYR61 were measured by quantitative RT-PCR and normalized to GAPDH control. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


E . Osmotic stress decreases YAP and 14 - 3 - 3 interaction . HEK293A cells were transiently transfected with Flag - YAP and Myc - 14 - 3 - 3 , and were serum starved for 16 hr . Cells were then treated with 0 . 2 M sorbitol for the indicated time points . Myc - 14 - 3 - 3 was immunoprecipitated and the associated Flag - YAP was detected by Western . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

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

E. Osmotic stress decreases YAP and 14-3-3 interaction. HEK293A cells were transiently transfected with Flag-YAP and Myc-14-3-3, and were serum starved for 16 hr. Cells were then treated with 0.2 M sorbitol for the indicated time points. Myc-14-3-3 was immunoprecipitated and the associated Flag-YAP was detected by Western. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


A . Inhibition of p38 or JNK does not block osmotic stress - induced YAP nuclear localization . HEK293A cells were pretreated with 2 μM p38 inhibitor ( SB203580 ) or 20 μM JNK inhibitor ( SP600125 ) before treatment with 0 . 2 M sorbitol for 1 hr in the absence of serum . Quantification of YAP / TAZ subcellular staining is shown . Scale bar : 20 μm .

O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

A. Inhibition of p38 or JNK does not block osmotic stress-induced YAP nuclear localization. HEK293A cells were pretreated with 2 μM p38 inhibitor (SB203580) or 20 μM JNK inhibitor (SP600125) before treatment with 0.2 M sorbitol for 1 hr in the absence of serum. Quantification of YAP/TAZ subcellular staining is shown. Scale bar: 20 μm.


A . Inhibition of p38 or JNK does not block osmotic stress - induced YAP nuclear localization . HEK293A cells were pretreated with 2 μM p38 inhibitor ( SB203580 ) or 20 μM JNK inhibitor ( SP600125 ) before treatment with 0 . 2 M sorbitol for 1 hr in the absence of serum . Cell lysates from identically treated samples were examined for phosphorylation of p38 substrate MK - 2 and JNK substrate c - Jun .

O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SMALL_MOLECULE 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 B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

A. Inhibition of p38 or JNK does not block osmotic stress-induced YAP nuclear localization. HEK293A cells were pretreated with 2 μM p38 inhibitor (SB203580) or 20 μM JNK inhibitor (SP600125) before treatment with 0.2 M sorbitol for 1 hr in the absence of serum. Cell lysates from identically treated samples were examined for phosphorylation of p38 substrate MK-2 and JNK substrate c-Jun.


B . NLK knockout blocks osmotic stress - induced YAP nuclear localization . HEK293A cells were transiently transfected with CRISPR / Cas9 to knockout NLK . Wild type ( WT ) cells and the NLK KO cell pool were treated with 0 . 2 M sorbitol for 1 hr in the absence of serum . YAP / TAZ subcellular localization was determined by immunofluorescence staining . Scale bar : 20 μm .

O O B-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_LINE 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 O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

B. NLK knockout blocks osmotic stress-induced YAP nuclear localization. HEK293A cells were transiently transfected with CRISPR/Cas9 to knockout NLK. Wild type (WT) cells and the NLK KO cell pool were treated with 0.2 M sorbitol for 1 hr in the absence of serum. YAP/TAZ subcellular localization was determined by immunofluorescence staining. Scale bar: 20 μm.


C . NLK induces YAP nuclear translocation . HEK293A cells were co - transfected with Flag - YAP together with vector control , Myc - NLK - WT ( wild type NLK ) , or NLK - KN ( kinase negative mutant ) . Cells were serum starved for 6 hr , and YAP localization and NLK expression were determined by Flag ( green ) and Myc ( red ) antibodies , respectively . Scale bar : 20 μm .

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

C. NLK induces YAP nuclear translocation. HEK293A cells were co-transfected with Flag-YAP together with vector control, Myc-NLK-WT (wild type NLK), or NLK-KN (kinase negative mutant). Cells were serum starved for 6 hr, and YAP localization and NLK expression were determined by Flag (green) and Myc (red) antibodies, respectively. Scale bar: 20 μm.


D . NLK induces YAP phosphorylation . HA - YAP was co - transfected with NLK - WT or NLK - KN in HEK293A cells . The phos - tag gel showed NLK - WT but not NLK - KN caused a significant mobility shift of YAP .

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

D. NLK induces YAP phosphorylation. HA-YAP was co-transfected with NLK-WT or NLK-KN in HEK293A cells. The phos-tag gel showed NLK-WT but not NLK-KN caused a significant mobility shift of YAP.


E . NLK phosphorylates YAP in vitro . NLK - WT and NLK - KN were immunoprecipitated from HEK293A cells and an in vitro kinase assay was performed using recombinant GST - YAP as a substrate in the presence of ATP - γ - S . Total phosphorylation of YAP was detected by immunoblotting with thiophosphate ester antibody , which identifies the alkylated thiophosphorylation on YAP .

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

E. NLK phosphorylates YAP in vitro. NLK-WT and NLK-KN were immunoprecipitated from HEK293A cells and an in vitro kinase assay was performed using recombinant GST-YAP as a substrate in the presence of ATP-γ-S. Total phosphorylation of YAP was detected by immunoblotting with thiophosphate ester antibody, which identifies the alkylated thiophosphorylation on YAP.


A . NLK induces YAP Ser 128 phosphorylation . Flag - YAP WT or S128A mutant was co - transfected with or without Myc - NLK . Phosphorylation was determined by Western blot using YAP S128 phosphospecific antibody .

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

A. NLK induces YAP Ser 128 phosphorylation. Flag-YAP WT or S128A mutant was co-transfected with or without Myc-NLK. Phosphorylation was determined by Western blot using YAP S128 phosphospecific antibody.


B . NLK phosphorylates YAP Ser 128 in vitro . NLK - WT or NLK - KN was transfected into HEK293A cells and immunoprecipitated . An in vitro NLK kinase assay was performed using recombinant GST - YAP as a substrate . Phosphorylation of YAP Ser 128 was determined by immunoblotting with phospho - YAP ( S128 ) antibody .

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

B. NLK phosphorylates YAP Ser 128 in vitro. NLK-WT or NLK-KN was transfected into HEK293A cells and immunoprecipitated. An in vitro NLK kinase assay was performed using recombinant GST-YAP as a substrate. Phosphorylation of YAP Ser 128 was determined by immunoblotting with phospho-YAP (S128) antibody.


C . Osmotic stress induces YAP phosphorylation at Ser 128 . In the left panel , HEK293A cells were treated with sorbitol and endogenous YAP was immunoprecipitated . In the right panel , Flag - YAP was transfected into HEK293A cells . Flag - YAP was immunoprecipitated , YAP Ser 128 phosphorylation was detected by a phosphospecific antibody . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

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

C. Osmotic stress induces YAP phosphorylation at Ser 128. In the left panel, HEK293A cells were treated with sorbitol and endogenous YAP was immunoprecipitated. In the right panel, Flag-YAP was transfected into HEK293A cells. Flag-YAP was immunoprecipitated, YAP Ser 128 phosphorylation was detected by a phosphospecific antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


D . NLK deficiency reduces YAP S128 phosphorylation . Two CRISPR / CAS9 gRNA plasmids targeting different sites of NLK were transfected into HEK293A . WT cells and two pools of NLK CRISPR / CAS9 transfected cells were treated with 0 . 2 M sorbitol for the indicated time points . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 4 ) .

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

D. NLK deficiency reduces YAP S128 phosphorylation. Two CRISPR/CAS9 gRNA plasmids targeting different sites of NLK were transfected into HEK293A. WT cells and two pools of NLK CRISPR/CAS9 transfected cells were treated with 0.2 M sorbitol for the indicated time points. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 4).


E . The S128D phosphomimetic mutant abolishes YAP interaction with 14 - 3 - 3 . Flag - YAP WT and mutant constructs were co - transfected with Myc - 14 - 3 - 3 into HEK293A cells . Cells were serum starved for 16 hrs . 14 - 3 - 3 was immunoprecipitated with Myc antibody and the associated YAP was detected with Flag antibody .

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

E. The S128D phosphomimetic mutant abolishes YAP interaction with 14-3-3. Flag-YAP WT and mutant constructs were co-transfected with Myc-14-3-3 into HEK293A cells. Cells were serum starved for 16 hrs. 14-3-3 was immunoprecipitated with Myc antibody and the associated YAP was detected with Flag antibody.


F . YAP Ser 128 phosphorylation is required for disruption of YAP - 14 - 3 - 3 interaction by osmotic stress . Flag - YAP WT or Flag - YAP S128A constructs were co - transfected with Myc - 14 - 3 - 3 into HEK293A cells . Cells were serum starved for 16 hrs , and treated with 0 . 2 M sorbitol for the indicated time points or refreshed with serum containing medium for 1 hr . Refreshing medium served as a control to disrupt YAP and 14 - 3 - 3 binding . 14 - 3 - 3 was immunoprecipitated with Myc antibody , and the associated YAP was detected with Flag antibody . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

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

F. YAP Ser 128 phosphorylation is required for disruption of YAP-14-3-3 interaction by osmotic stress. Flag-YAP WT or Flag-YAP S128A constructs were co-transfected with Myc-14-3-3 into HEK293A cells. Cells were serum starved for 16 hrs, and treated with 0.2 M sorbitol for the indicated time points or refreshed with serum containing medium for 1 hr. Refreshing medium served as a control to disrupt YAP and 14-3-3 binding.14-3-3 was immunoprecipitated with Myc antibody, and the associated YAP was detected with Flag antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


A . NLK does not induce nuclear translocation of the S128A mutant YAP . HEK293A cells were co - transfected with Myc - NLK and Flag - YAP WT or S128A mutant . Cells were serum starved for 6 hr , and YAP localization and NLK expression were determined by Flag ( green ) and Myc ( red ) antibodies , respectively . Scale bar : 20 μm .

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

A. NLK does not induce nuclear translocation of the S128A mutant YAP. HEK293A cells were co-transfected with Myc-NLK and Flag-YAP WT or S128A mutant. Cells were serum starved for 6 hr, and YAP localization and NLK expression were determined by Flag (green) and Myc (red) antibodies, respectively. Scale bar: 20 μm.


B . YAP Ser 128 phosphorylation is required for osmotic stress - induced YAP nuclear localization . HEK293A YAP KO cells reconstituted with Flag - YAP WT , YAP S128D , or YAP S128A mutant were treated with 0 . 2 M sorbitol for 1 hr . YAP subcellular localization was determined by Flag immunofluorescence staining ( green ) . Scale bar : 20 μm .

O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-CELL_LINE B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O

B. YAP Ser 128 phosphorylation is required for osmotic stress-induced YAP nuclear localization. HEK293A YAP KO cells reconstituted with Flag-YAP WT, YAP S128D, or YAP S128A mutant were treated with 0.2 M sorbitol for 1 hr. YAP subcellular localization was determined by Flag immunofluorescence staining (green). Scale bar: 20 μm.


A . YAP S128D reconstituted cells show growth advantage under hyperosmotic conditions . YAP / TAZ dKO HEK293A cells with stable expression of YAP WT , S128A , and S128D were cultured in the absence ( left panel ) or presence of 0 . 2M sorbitol ( right panel ) for the indicated amount of time . Cell numbers were counted and normalized to day 0 . Data are presented as mean ± SEM , n = 3 .

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

A. YAP S128D reconstituted cells show growth advantage under hyperosmotic conditions. YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D were cultured in the absence (left panel) or presence of 0.2M sorbitol (right panel) for the indicated amount of time. Cell numbers were counted and normalized to day 0. Data are presented as mean ± SEM, n = 3.


B . YAP S128D reconstituted cells have lower cell death in a hyperosmotic environment . Cell cycle analyses of YAP / TAZ dKO HEK293A cells with stable expression of YAP WT , S128A , and S128D after 0 . 2 M sorbitol treatment were determined using flow cytometry . Propidium iodine ( PI ) was used for DNA staining ( Fig EV5 ) . Quantification of sub - G1 cells is shown . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

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

B. YAP S128D reconstituted cells have lower cell death in a hyperosmotic environment. Cell cycle analyses of YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D after 0.2 M sorbitol treatment were determined using flow cytometry. Propidium iodine (PI) was used for DNA staining (Fig EV5). Quantification of sub-G1 cells is shown. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


C . YAP S128D reconstituted cells show reduced apoptosis under hyperosmotic conditions . Annexin V analyses of YAP / TAZ dKO HEK293A cells with stable expression of YAP WT , S128A , and S128D after 0 . 2 M sorbitol treatment were determined using flow cytometry . PE - Annexin V and 7 - AAD stained for phospholipid phosphatidylserine ( PS ) and DNA , respectively . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

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

C. YAP S128D reconstituted cells show reduced apoptosis under hyperosmotic conditions. Annexin V analyses of YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D after 0.2 M sorbitol treatment were determined using flow cytometry. PE-Annexin V and 7-AAD stained for phospholipid phosphatidylserine (PS) and DNA, respectively. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


G , Benzidine staining of whole spleen [ top ] or marrow [ bottom ] from 3 month old mice [ left ] or 3 year old naked mole - rats [ right ] . Scale bar 250µm , arrows indicate nucleated erythroid progenitors ( NEPs ) .

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

G, Benzidine staining of whole spleen [top] or marrow [bottom] from 3 month old mice [left] or 3 year old naked mole-rats [right]. Scale bar 250µm, arrows indicate nucleated erythroid progenitors (NEPs).


H , Relative counts of Benzidine - stained cytospins from whole spleen or WBM . p - value determined by Sidak ' s Two - way ANOVA comparing BM vs spleen between mouse ( n = 5 ) and naked mole - rat ( n = 8 ) . I , Relative counts of Benzidine - stained cytospins from naked mole - rat sorted spleen fractions ; p - value determined by Sidak ' s Two - way ANOVA comparing BM vs spleen ; n = 4 .

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

H, Relative counts of Benzidine-stained cytospins from whole spleen or WBM. p-value determined by Sidak's Two-way ANOVA comparing BM vs spleen between mouse (n=5) and naked mole-rat (n=8). I, Relative counts of Benzidine-stained cytospins from naked mole-rat sorted spleen fractions; p-value determined by Sidak's Two-way ANOVA comparing BM vs spleen; n=4.


A , Cross - species integration of bulk RNA - Seq datasets . Naked mole - rat BM populations CP1 - 4 were matched to human and murine counterparts for GSEA with MSigDB hallmark genesets . Primitive stem and progenitor [ left ] , hsa : LT - HSC , LIN - / CD34 + / CD38lo / CD90 + / CD45RA - ( n = 7 ) ; HSPC , LIN - / CD34 + / CD38lo ( n = 11 ) ; MPP , LIN - / CD34 + / CD38lo / CD90 - / CD45RA - ( n = 4 ) ; mmu : LT - HSC , LIN - / Sca - 1 + / Kit + / CD150 + / CD48 - ( n = 25 ) ; MPP , LIN - / Sca - 1 + / Kit + / CD150 - / CD48 + ( n = 6 ) ; hgl : LTC , LIN - / Thy1 . 1int / CD34 + ( n = 5 ) . Lymphomyeloid [ center ] , hsa : LMPP , LIN - / CD34 + / CD38lo / CD123lo / CD45RA + ( n = 9 ) ; CLP , LIN - / CD34 + / CD38hi / CD10 + / CD45RA + ( n = 6 ) ; MLP , LIN - / CD34 + / CD38lo / CD90 - / CD45RA + / CD71 - ( n = 4 ) ; CMP , LIN - / CD34 + / CD38hi / CD123lo / CD45RA - ( n = 26 ) ; GMP , LIN - / CD34 + / CD38lo / CD123lo / CD45RA + ( n = 18 ) ; mmu : LMPP , LIN - / Sca - 1 + / Kit + / Flt3hi ( n = 5 ) ; CMP , LIN - / Sca - 1 - / Kit + / CD16 / 32lo / CD34 + ( n = 8 ) ; GMP , LIN - / Sca - 1 - / Kit + / CD16 / 32hi / CD34 + ( n = 16 ) ; hgl : CP1 , LIN + / Thy1 . 1int / CD34 + ( n = 5 ) . Erythroid [ right ] , hsa : MEP , LIN - / CD34 + / CD38hi / CD123 - / CD45RA - ( n = 23 ) ; MKP , LIN - / CD41a + / CD42b + ( n = 3 ) ; ERY , LIN - / CD34lo / - / CD36 + / GYPA + / CD71 + ( n = 12 ) ; mmu : MEP , LIN - / Sca - 1 - / Kit + / CD16 / 32 - / CD34 - ( n = 15 ) ; hgl : CP3 , LIN - / Thy1 . 1lo / CD34 + ( n = 5 ) ; CP4 , LIN - / Thy1 . 1 - / CD34 + ( n = 5 ) . Within each group , cell types were pooled for each species . Shown are pathways related to proliferation and metabolism normalized enrichment score ; GeneRatio , ( signature ∩ term ) / ( signature ∩ all terms ) ; FDR , false discovery rate .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-ORGANISM O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O B-ORGANISM O 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 B-ORGANISM O B-CELL_TYPE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-ORGANISM O 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 B-ORGANISM O B-CELL_TYPE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O B-ORGANISM O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-ORGANISM O 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 B-GENEPROD I-GENEPROD I-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 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, Cross-species integration of bulk RNA-Seq datasets. Naked mole-rat BM populations CP1-4 were matched to human and murine counterparts for GSEA with MSigDB hallmark genesets. Primitive stem and progenitor [left], hsa: LT-HSC, LIN-/CD34+/CD38lo/CD90+/CD45RA- (n=7); HSPC, LIN-/CD34+/CD38lo (n=11); MPP, LIN-/CD34+/CD38lo/CD90-/CD45RA- (n=4); mmu: LT-HSC, LIN-/Sca-1+/Kit+/CD150+/CD48- (n=25); MPP, LIN-/Sca-1+/Kit+/CD150-/CD48+ (n=6); hgl: LTC, LIN-/Thy1.1int/CD34+ (n=5). Lymphomyeloid [center], hsa: LMPP, LIN-/CD34+/CD38lo/CD123lo/CD45RA+ (n=9); CLP, LIN-/CD34+/CD38hi/CD10+/CD45RA+ (n=6); MLP, LIN-/CD34+/CD38lo/CD90-/CD45RA+/CD71- (n=4); CMP, LIN-/CD34+/CD38hi/CD123lo/CD45RA- (n=26); GMP, LIN-/CD34+/CD38lo/CD123lo/CD45RA+ (n=18); mmu: LMPP, LIN-/Sca-1+/Kit+/Flt3hi (n=5); CMP, LIN-/Sca-1-/Kit+/CD16/32lo/CD34+ (n=8); GMP, LIN-/Sca-1-/Kit+/CD16/32hi/CD34+ (n=16); hgl: CP1, LIN+/Thy1.1int/CD34+ (n=5). Erythroid [right], hsa: MEP, LIN-/CD34+/CD38hi/CD123-/CD45RA- (n=23); MKP, LIN-/CD41a+/CD42b+ (n=3); ERY, LIN-/CD34lo/-/CD36+/GYPA+/CD71+ (n=12); mmu: MEP, LIN-/Sca-1-/Kit+/CD16/32-/CD34- (n=15); hgl: CP3, LIN-/Thy1.1lo/CD34+ (n=5); CP4, LIN-/Thy1.1-/CD34+ (n=5). Within each group, cell types were pooled for each species. Shown are pathways related to proliferation and metabolism normalized enrichment score; GeneRatio, (signature ∩ term) / (signature ∩ all terms); FDR, false discovery rate.


B , Seahorse assay with cell types sorted according to ( A ) ; hOPP , human oligopotent progenitors LIN - / CD34 + / CD38 + ; mOPP , LIN - / Sca - 1 - / Kit + . ECAR , extracellular acidification rate ; OCR , oxygen consumption rate . Error bars , s . e . m . ; Human cell types , n = 4 ; mouse , n = 3 , naked mole - rat , n = 3 .

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

B, Seahorse assay with cell types sorted according to (A); hOPP, human oligopotent progenitors LIN-/CD34+/CD38+; mOPP, LIN-/Sca-1-/Kit+. ECAR, extracellular acidification rate; OCR, oxygen consumption rate. Error bars, s.e.m.; Human cell types, n=4; mouse, n=3, naked mole-rat, n=3.


C , Mitotracker Red staining in mouse ( n = 4 ) , human ( n = 4 ) and naked mole - rat ( n = 5 ) BM , histogram of merged per - species data . unstained , BM from mouse [ solid ] , human [ dotted ] or NMR [ area under curve ( AUC ) ] ; yLSK , LIN - / Sca - 1 + / Kit + , 3 month old ; oLSK , 24 month ; yLTC , LIN - / Thy1 . 1int / CD34 + , 3 year old ; hHSC , human CD34 + / CD38lo ;

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

C, Mitotracker Red staining in mouse (n=4), human (n=4) and naked mole-rat (n=5) BM, histogram of merged per-species data. unstained, BM from mouse [solid], human [dotted] or NMR [area under curve (AUC)]; yLSK, LIN-/Sca-1+/Kit+, 3 month old; oLSK, 24 month; yLTC, LIN-/Thy1.1int/CD34+, 3 year old; hHSC, human CD34+/CD38lo;


F , Confocal fluorescence imaging of sorted mouse LT - HSCs ( LIN - / Sca - 1 + / Kit + / CD150 + / CD48 - ) and NMR LTCs across age ; Y , young ( mouse , 4 month - old ; NMR , 3 year - old ) ; O , old ( mouse , 24 month - old ) ; MA , middle - aged ( NMR , 11 year - old ) . Scale bar 10µm .

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

F, Confocal fluorescence imaging of sorted mouse LT-HSCs (LIN-/Sca-1+/Kit+/CD150+/CD48-) and NMR LTCs across age; Y, young (mouse, 4 month-old; NMR, 3 year-old); O, old (mouse, 24 month-old); MA, middle-aged (NMR, 11 year-old). Scale bar 10µm.


G , H Polarization quantification for G , mouse LT - HSC ( biological n = 3 ) ; and H , NMR LTC ( Y , n = 5 ; MA , n = 4 ) . Line at mean ; error bars , s . e . m . ; p - value determined by Sidak ' s Two - way ANOVA .

O O O B-EXP_ASSAY O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 O O O O O O O O O O O O O O O O O O O

G, H Polarization quantification for G, mouse LT-HSC (biological n=3); and H, NMR LTC (Y, n=5; MA, n=4). Line at mean; error bars, s.e.m.; p-value determined by Sidak's Two-way ANOVA.


A U2OS cells were transfected with Flag - NAT10 or control vectors . Forty - eight hours later , cells were harvested and whole cell extracts were immunoprecipitated with Flag - antibody affinity resin . The NAT10 - binding proteins were resolved by SDS - PAGE and detected by silver staining .

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

A U2OS cells were transfected with Flag-NAT10 or control vectors. Forty-eight hours later, cells were harvested and whole cell extracts were immunoprecipitated with Flag-antibody affinity resin. The NAT10-binding proteins were resolved by SDS-PAGE and detected by silver staining.


B - F U2OS , HCT116 p53 + / + or HCT116 p53 - / - cell lysates were immunoprecipitated with control IgG , anti - NAT10 ( B and F ) , anti - p53 ( C ) and anti - Mdm2 ( D and E ) antibodies . The immunoprecipitates were subsequently immunoblotted with the indicated antibodies .

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

B-F U2OS, HCT116 p53+/+ or HCT116 p53-/- cell lysates were immunoprecipitated with control IgG, anti-NAT10 (B and F), anti-p53 (C) and anti-Mdm2 (D and E) antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies.


G Purified NAT10 was incubated with GST , GST - p53 or GST - Mdm2 proteins coupled to Glutathione Sepharose 4B . Proteins retained on the Sepharose were then analyzed by western blot using the antibodies as indicated . The amount of GST fusion proteins are shown in the lower panel .

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

G Purified NAT10 was incubated with GST, GST-p53 or GST-Mdm2 proteins coupled to Glutathione Sepharose 4B. Proteins retained on the Sepharose were then analyzed by western blot using the antibodies as indicated. The amount of GST fusion proteins are shown in the lower panel.


H Full - length GST - p53 fusion protein , its deletion mutants or GST protein was used to pull - down experiments with purified NAT10 protein . The levels of the GST fusion proteins are shown in the left panel .

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

H Full-length GST-p53 fusion protein, its deletion mutants or GST protein was used to pull-down experiments with purified NAT10 protein. The levels of the GST fusion proteins are shown in the left panel.


I GST pull - down assay was performed using purified GST - NAT10 deletion mutants or GST protein and overexpressed Flag - p53 or Mdm2 protein in HEK293T cells . Schematic diagram represents the constructs of GST - NAT10 deletion mutants ( right panel ) .

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

I GST pull-down assay was performed using purified GST-NAT10 deletion mutants or GST protein and overexpressed Flag-p53 or Mdm2 protein in HEK293T cells. Schematic diagram represents the constructs of GST-NAT10 deletion mutants (right panel).


A Recombinant His - p53 was incubated with or without purified Flag - NAT10 as described in Experimental Procedures . The reaction products were resolved by SDS - PAGE and immunoblotted using anti - acetyl - lysine antibodies . Flag - PCAF was used as a positive control .

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

A Recombinant His-p53 was incubated with or without purified Flag-NAT10 as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE and immunoblotted using anti-acetyl-lysine antibodies. Flag-PCAF was used as a positive control.


B In vitro acetylation was performed and the acetylated p53 was detected as in A .

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

B In vitro acetylation was performed and the acetylated p53 was detected as in A.


C In vitro acetylation assay was performed as described in A . The reaction products were resolved by SDS - PAGE and acetylated p53 was purified from the SDS - PAGE and subjected to mass spectrometry analysis .

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

C In vitro acetylation assay was performed as described in A. The reaction products were resolved by SDS-PAGE and acetylated p53 was purified from the SDS-PAGE and subjected to mass spectrometry analysis.