( D ) Sucrose density gradient sedimentation profile of ribosomal mtRNAs from heartmitochondria . migration of ribosomal subunits ( 28S 39S ) mitoribosome ( 55S ) was detected by immunobloting with antibodies against MRPS35 MRPL12 . profile of a given mt - is calculated after qRT - PCR of fractions corrected for its abundance in according to results of Northern blot . Results of experiments are provided for 12S 16S rRNA , for mt - mRNAs , results of distribution from two independent experiments using probes against ATP6 , COXIII ND5 were combined . Error bars represent mean ± SD . Student ' s t - was used to determine level of statistical difference ( n = 3 ) .

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(D) Sucrose density gradient sedimentation profile of ribosomal proteins and mtRNAs from heartmitochondria. The migration of ribosomal subunits (28S and 39S) and mitoribosome (55S) was detected by immunobloting with antibodies against MRPS35 and MRPL12. The profile of a given mt-RNA is calculated after qRT-PCR analysis of individual fractions and corrected for its abundance in the cell according to results of Northern blot analysis. Results of individual experiments are provided for 12S and 16S rRNA, and for mt-mRNAs, results of distribution from two independent experiments using probes against ATP6, COXIII and ND5 were combined. Error bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=3).


( E ) Relative association of 12S rRNA , 16S rRNA mt - mRNAs with mitoribosomes ( 55S ) in Clpp knockout mitochondria comparing to wild type . Bars represent mean ± SD . Student ' s t - was used to determine level of statistical difference ( 12S 16S n = 3 ; mRNA n = 6 ) .

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(E) Relative association of 12S rRNA, 16S rRNA and mt-mRNAs with mitoribosomes (55S) in Clpp knockout mitochondria comparing to wild type. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S and 16S n=3; mRNA n=6).


( B ) Western blot subsequent of ERAL1 EFG1 from Clpp knockout ( Clpp - / - ) heartmitochondria relative to wild type ( Clpp + / + ) . Bars represent mean ± SD . Student ' s t - was used to determine level of statistical difference ( n = 4 ) . arrowhead indicates positions of EFG1 .

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(B) Western blot analysis and subsequent quantification of ERAL1 and EFG1 levels from Clpp knockout (Clpp-/-) heartmitochondria relative to wild type (Clpp+/+). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=4). The arrowhead indicates positions of EFG1.


( C ) Expression of potential CLPP substrates from Clpp knockout ( Clpp - / - ) heartmitochondria relative to wild type ( set to 1 ) . Bars represent mean ± SD . Student ' s t - was used to determine level of statistical difference ( n = 4 ) .

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(C) Expression levels of potential CLPP substrates from Clpp knockout (Clpp-/-) heartmitochondria relative to wild type (set to 1). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=4).


( D ) Cycloheximide ( CHX ) chase to determine stability of ERAL1 EFG1 . are visualized by Western blot with respective antibodies . are isolated after 2 , 4 , 6 , 8 , 10 12 hours after beginning of chase ( 0 ) . arrowhead indicates positions of ERAL1 .

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(D) Cycloheximide (CHX) chase assay to determine the stability of ERAL1 and EFG1. Proteins are visualized by Western blot with respective antibodies. Proteins are isolated after 2, 4, 6, 8,10 and 12 hours after beginning of chase (0). The arrowhead indicates positions of ERAL1.


( E ) Western blot subsequent of in wild type ( C1 , C2 ) Clpp knockout ( KO1 , KO2 ) MEFs upon Clpx siRNA ( Clpx ) relative to untreated ( Ctrl ) wild type . Bars represent mean ± SD . Student ' s t - was used to determine level of statistical difference ( n = 6 ) . arrowhead indicates positions of either CLPX or ERAL1 , stars indicate position of cross - reacting bands .

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(E) Western blot analysis and subsequent quantification of protein levels in wild type (C1, C2) and Clpp knockout (KO1, KO2) MEFs upon Clpx siRNA (Clpx) relative to untreated (Ctrl) wild type. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (n=6). The arrowhead indicates positions of either CLPX or ERAL1, and stars indicate position of cross-reacting bands.


( A ) Sucrose density gradient sedimentation profile ( " short gradients " ) of ribosomal transcripts from control ( Clpp + / + ) Clpp knockout ( Clpp - / - ) heartmitochondria . migration of ribosomal subunits ( 28S 39S ) mitoribosome ( 55S ) was detected by immunoblotting with antibodies against MRPS35 MRPL12 . 12S rRNA was analysed by Northern blot .

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(A) Sucrose density gradient sedimentation profile ("short gradients") of ribosomal proteins and transcripts from control (Clpp+/+) and Clpp knockout (Clpp-/-) heartmitochondria. The migration of ribosomal subunits (28S and 39S) and mitoribosome (55S) was detected by immunoblotting with antibodies against MRPS35 and MRPL12. 12S rRNA was analysed by Northern blot.


( B ) Sedimentation profiles of ERAL1 , MRPS28 , EFG1 , CLPP , CLPX , EFTU MTIF2 in heartmitochondria as in A .

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(B) Sedimentation profiles of ERAL1, MRPS28, EFG1, CLPP, CLPX, EFTU and MTIF2 in heartmitochondria as in A.


( C ) Relative abundance of bound to 28S ribosomal subunit ( fractions 3 up ) . Bars represent mean ± SD . Student ' s t - was used to determine level of statistical difference ( ERAL1 n = 4 ; EFG1 , CLPX , MTIF2 n = 3 ; EFTU n = 2 ) .

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(C) Relative abundance of proteins bound to the 28S ribosomal subunit (fractions 3 and up). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (ERAL1 n=4; EFG1, CLPX, MTIF2 n=3; EFTU n=2).


( D ) Sucrose density gradient sedimentation profile ( " long gradients " as in figure 4D ) of ERAL1 , MRPS35 , MRPL37 , CLPX EFG1 from control ( Clpp + / + ) Clpp knockout ( Clpp - / - ) heartmitochondria . same amount of lysates were separated by gradient amount of each is estimated after Western blot ( Input ) . Only fractions 4 - 11 containing 28S , 39S 55S ribosomal fractions were blotted to avoid high signals arising in free fractions ( 1 - 3 ) .

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(D) Sucrose density gradient sedimentation profile ("long gradients" as in figure 4D) of ERAL1, MRPS35, MRPL37, CLPX and EFG1 from control (Clpp+/+) and Clpp knockout (Clpp-/-) heartmitochondria. The same amount of protein lysates were separated by the gradient and the amount of each individual protein is estimated after Western blot analysis (Input). Only fractions 4 - 11 containing the 28S, 39S and 55S ribosomal fractions were blotted to avoid high signals arising in free fractions (1-3).


( A ) Coimmunoprecipitation using antibodies raised against ERAL1 CLPX . Western blot of input ( I ) , control ( C ) eluate ( E ) is shown . Asterix indicates cross - reacting band .

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(A) Coimmunoprecipitation analysis using antibodies raised against ERAL1 and CLPX proteins. Western blot analysis of input (I), control (C) and eluate (E) is shown. Asterix indicates the cross-reacting band.


( B ) Sucrose density gradient migration profile - association of ERAL1 with 28S ribosomal subunit ( MRPS35 ) upon Eral1 siRNA . TOMM20 was used as a loading control .

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(B) Sucrose density gradient migration profile - association of ERAL1 with the 28S ribosomal subunit (MRPS35) upon Eral1 siRNA. TOMM20 was used as a loading control.


( C ) De novo synthesis of mitochondrial in wild type ( Clpp + / + ) Clpp knockout ( Clpp - / - ) MEFs upon Eral1 siRNA . were isolated after labeling with 35S - methionine for 1 hour from on control siRNA ( Ctrl ) or on Eral1 siRNA ( Eral1 ) . Positions of mitochondrial encoded are indicated . Coomassie blue was used to ensure equal loading .

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(C) De novo synthesis of mitochondrial proteins in wild type (Clpp+/+) and Clpp knockout (Clpp-/-) MEFs upon Eral1 siRNA. Proteins were isolated after labeling with 35S-methionine for 1 hour from cells on control siRNA (Ctrl) or cells on Eral1 siRNA (Eral1). Positions of individual mitochondrial encoded proteins are indicated. Coomassie blue staining was used to ensure equal loading.


( D ) of Western blot of respiratory chain subunits upon knockdown of Eral1 . relative to wild type treated with control siRNA ( set to 1 ) are presented . Bars represent mean ± SD . Student ' s t - was used to determine level of statistical difference ( COXI n = 4 ; ATP5A1 n = 2 ; n = 6 ) . Combined results from 2 - 3 blots were used for as only 2 samples of each conditions are used in a single experiment . Numbers within bars represent statistical difference to control wild type numbers above bars represent difference in Clpp - knockout upon Eral1 siRNA .

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(D) Quantification of Western blot analysis of respiratory chain subunits upon knockdown of Eral1. Levels relative to wild type cells treated with control siRNA (set to 1) are presented. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (COXI n=4; ATP5A1 n=2; the rest n=6). Combined results from 2-3 blots were used for quantification as only 2 samples of each conditions are used in a single experiment. Numbers within bars represent statistical difference to control wild type cells and numbers above bars represent difference in Clpp-knockout cells upon Eral1 siRNA.


( E ) Western blot of RC subunits upon Eral1 knockdown in MEFs . ( 0 ) were treated with doxycycline for 48 hours ( T ) upon doxycycline removal allowed to recover for 24 hours ( R ) . ( F ) of relative recovery of COXI NDUFB6 upon Eral1 siRNA . values are calculated as between recovered ( R ) to doxycycline untreated ( 0 ) in Eral1 - knockdown ( Eral1 ) compared to same results obtained in control ( Ctrl ) . Bars represent mean ± SD . Student ' s t - was used to determine level of statistical difference ( COXI n = 4 ; NDUFB6 n = 3 ) . Numbers within bars represent statistical difference between control / Eral1 siRNA treated numbers above bars represent difference between + / + ; Eral1 siRNA - / - ; Eral1 siRNA .

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(E) Western blot analysis of RC subunits upon Eral1 knockdown in MEFs. Cells (0) were treated with doxycycline for 48 hours (T) and upon doxycycline removal allowed to recover for 24 hours (R).(F) Quantification of the relative recovery of COXI and NDUFB6 upon Eral1 siRNA. The values are calculated as ratio between recovered (R) to doxycycline untreated (0) protein levels in Eral1-knockdown cells (Eral1) and compared to the same results obtained in control cells (Ctrl). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (COXI n=4; NDUFB6 n=3). Numbers within bars represent statistical difference between control/Eral1 siRNA treated cells and numbers above bars represent difference between +/+; Eral1 siRNA and -/-; Eral1 siRNA.


B . ERK71 mutants have elevated triacylglycerol ( TAG ) ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) . C . Fat specific depletion of ERK7 by RNAi ( BDSC 56939 ) leads to increased TAG ( N = 4 replicates of 10 larvae / replicate for each genotype ) .

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B. ERK71 mutants have elevated triacylglycerol (TAG) levels (N=4 replicates of ≥10 larvae/ replicate for each genotype). C. Fat body specific depletion of ERK7 by RNAi (BDSC 56939) leads to increased TAG levels (N=4 replicates of 10 larvae/replicate for each genotype).


D . Representative immunofluorescent of lipid droplet ( LipidTOX ) ( DAPI ) in control ERK71 mutant fat bodies of third instar larvae . Scale bar : 50 µm . E . ERK71 mutant fat contain more lipid droplets than control ( N = 30 for each genotype ) .

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D. Representative immunofluorescent images of lipid droplet (LipidTOX) and nuclear (DAPI) staining in control and ERK71 mutant fat bodies of third instar larvae. Scale bar: 50 µm. E. ERK71 mutant fat body cells contain more lipid droplets than control cells (N=30 cells for each genotype).


F . After fed [ 13C ] glucose , ERK71 mutant fat bodies display elevated [ 13C ] TAG / PE unlabeled TAG / PE molar ratios ( measured by mass spectrometry - lipidomics , N = 3 replicates of 15 fat bodies / replicate , TAG = triacylglycerol , PE = phosphatidylethanolamine )

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F. After fed [13C]glucose, the ERK71 mutant fat bodies display elevated [13C]TAG/PE and unlabeled TAG/PE molar ratios (measured by mass spectrometry-based lipidomics, N=3 replicates of 15 fat bodies/replicate, TAG=triacylglycerol, PE=phosphatidylethanolamine)


G . ERK7 overexpression in fat results in reduced TAG , while kinase - dead ( K54R ) activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence TAG storage ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) .

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G. ERK7 overexpression in the fat body results in reduced TAG levels, while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the TAG storage (N=4 replicates of ≥10 larvae/replicate for each genotype).


H - ERK7 expressing , GFP - marked , fat clone ( H , marked by yellow dotted ; scale bar : 50 µm ) visualized by LipidTOX ( N = 11 for control 4 for ERK7 overexpression ) .

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H- ERK7 expressing, GFP-marked, fat body clone (H, marked by yellow dotted line; scale bar: 50 µm) visualized by LipidTOX staining (N=11 for control and 4 for ERK7 overexpression).


ERK7 expressing , fat clone contains less ( I ) smaller ( J ) lipid droplets than control ( N = 11 for control 4 for ERK7 overexpression ) .

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ERK7 expressing, fat body clone contains less (I) and smaller (J) lipid droplets than control cells (N=11 for control and 4 for ERK7 overexpression).


K . qRT - PCR expression of ERK7 from fat bodies of w1118 larvae upon 6 h starvation . Expression of RP49 was used for normalization ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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K. qRT-PCR based expression analysis of ERK7 from fat bodies of w1118 larvae upon 6 h starvation. Expression of RP49 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).


A . Fatty acid composition of total lipids in CG > control CG > ERK7 larvae , measured by chromatography ( N = 3 replicates of 15 larvae / replicate for each genotype ) .

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A. Fatty acid composition of total lipids in CG>control and CG>ERK7 larvae, measured by gas chromatography (N=3 replicates of 15 larvae/replicate for each genotype).


C . Amount of glucose - derived [ 13C ] - labeled carbons incorporated into TAG pool of control CG > ERK7 fat bodies ( N = 3 replicates of 15 fat bodies / replicate for each genotype diet ) .

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C. Amount of glucose-derived [13C]-labeled carbons incorporated into the TAG pool of control and CG>ERK7 fat bodies (N=3 replicates of 15 fat bodies/replicate for each genotype and diet).


D . Ectopic ERK7 expression in fat results in elevated hemolymph glucose ( N = 3 replicates of 15 larvae / replicate for each genotype ) . E . Circulating glucose are decreased in ERK71 mutants ( N = 4 replicates of 15 larvae / replicate for each genotype ) .

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D. Ectopic ERK7 expression in the fat body results in elevated hemolymph glucose levels (N=3 replicates of 15 larvae/replicate for each genotype). E. Circulating glucose levels are decreased in ERK71 mutants (N=4 replicates of 15 larvae/replicate for each genotype).


A . Four - way plot ( t - statistics ) presenting ERK7 - dependent fasting - responsive genes ( adj . p . val < 0 . 05 ) . blue indicates significant in both comparisons ; turquoise , significant in one comparison ; turquoise , not significant .

A. Four-way plot (t-statistics) presenting the ERK7-dependent fasting-responsive genes (adj.p.val < 0.05). Dark blue indicates significant in both comparisons; dark turquoise, significant in one comparison; light turquoise, not significant.


D . Heatmaps displaying expression patterns of selected genes involved in lipid anabolism lipid catabolism . Color key displays scaled log2 gene expression .

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D. Heatmaps displaying expression patterns of selected genes involved in lipid anabolism and lipid catabolism. Color key displays scaled log2 gene expression.


E , F . Expression of Seipin , Agpat3 , ATPCL , fabp , Lip4 CG34448 by qRT - PCR . RP49 was used for normalization ( N = 3 replicates of ≥ 10 larvae / replicate for each genotype ) .

E, F. Expression analysis of Seipin, Agpat3, ATPCL, fabp, Lip4 and CG34448 by qRT-PCR. RP49 was used for normalization (N=3 replicates of ≥10 larvae/replicate for each genotype).


A . of control ERK71 mutant larvae at 72h after egg deposition ( AED ) ( N = 4 replicates of 12 larvae / replicate for each genotype ) .

A. Weight of control and ERK71 mutant larvae at 72h after egg deposition (AED) (N=4 replicates of 12 larvae/replicate for each genotype).


B . Pupariation kinetics of control ERK71 mutants ( N = 4 replicates of 30 larvae / replicate for each genotype ) . p < 0 . 0001 , analyzed by Log - rank .

B. Pupariation kinetics of control and ERK71 mutants (N=4 replicates of 30 larvae/replicate for each genotype). p<0.0001, analyzed by Log-rank test.


C . ERK71 mutants fail to suppress growth upon 24 h of starvation ( N ≥ 4 replicates of 15 larvae / replicate for each genotype ) .

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C. ERK71 mutants fail to suppress growth upon 24 h of starvation (N≥4 replicates of 15 larvae/replicate for each genotype).


D . ERK71 mutant larvae ( N = 4 replicates of 24 larvae / replicate for each genotype , p < 0 . 01 analyzed by Log - rank ) are sensitive to nutrient deprivation .

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D. ERK71 mutant larvae (N=4 replicates of 24 larvae/replicate for each genotype, p<0.01 analyzed by Log-rank test) are sensitive to nutrient deprivation.


Fat specific knockdown of ERK7 by RNAi ( BDSC 56939 ) leads to a moderately accelerated pupariation rate ( E , p < 0 . 001 analyzed by Log - rank , N = 8 replicates of 30 larvae / replicate for each genotype )

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Fat body specific knockdown of ERK7 by RNAi (BDSC 56939) leads to a moderately accelerated pupariation rate (E, p<0.001 analyzed by Log-rank test, N=8 replicates of 30 larvae/replicate for each genotype)


F . Fat specific knockdown of ERK7 by RNAi ( BDSC 56939 ) leads to an increase in larval at 72h AED ( F , N = 5 replicates of 15 larvae / replicate for each genotype ) .

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F. Fat body specific knockdown of ERK7 by RNAi (BDSC 56939) leads to an increase in larval weight at 72h AED (F, N=5 replicates of 15 larvae/replicate for each genotype).


G . A representative image showing that fat - specific overexpression of ERK7 by CG - GAL4 driver ( CG > ERK7 ) impairs larval growth . Scale bar : 0 . 5 mm .

G. A representative image showing that fat body-specific overexpression of ERK7 by the CG-GAL4 driver (CG>ERK7) impairs larval growth. Scale bar: 0.5 mm.


H . Ectopic expression of ERK7 leads to delayed pupariation ( p < 0 . 0001 , analyzed by Log - rank ) , while kinase - dead ( K54R ) activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence pupariation rate ( N = 4 replicates of 30 larvae / replicate for each genotype ) .

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H. Ectopic expression of ERK7 leads to delayed pupariation (p<0.0001, analyzed by Log-rank test), while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence pupariation rate (N=4 replicates of 30 larvae/replicate for each genotype).


I . In contrast to ERK7 wt , kinase - dead ( K54R ) activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence pupal volume ( N = 4 replicates of 10 pupae / replicate for each genotype ) .

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I. In contrast to ERK7 wt, kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the pupal volume (N=4 replicates of 10 pupae/replicate for each genotype).


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

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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 clones ( D , marked by GFP , scale bar : 20 µm ) display reduced ( E ) reduced nucleolar / ( F ) , when compared to control . Nuclei were visualized by DAPI , nucleoli were visualized by anti - Fibrillarin antibodies ( N = 10 ) .

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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 of Pol I ( G ) 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 ) .

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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 ( blue ) remain unresponsive to ERK7 .

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

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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 of PWP1 localization in fat bodies of third instar larvae . ERK7 overexpression ( GFP marked clones ) alters subcellular localization of PWP1 . Scale bar : 30 µm . E . PWP1 localization displays reduced / in ERK7 overexpressing clones , compared to control ( N = 10 from at least 8 different 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).


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

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

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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 ) ectopic expression of ERK7 ( B ) ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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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 ) .

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


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

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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 fat partially rescues TAG in CG > ERK7 larvae ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) .

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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 phenotype of ERK71 mutant fat ( N ≥ 25 per genotype ) .

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

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

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

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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 of Q97 - GFP . Bars , 5 µm . G . of containing or lacking detectable was quantified . boundaries are indicated by dashed lines .

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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 corresponding wild type for times indicated . were harvested , washed survivors were counted after plating on glucose plates . Mean values standard deviations of three replicates are shown .

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

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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 of indicated strains 12 h after shifting them to galactose . Note that in GAL - Mia40 form number of is very different to WT . Bar , 5 µm .

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

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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 in Q25 - GFP - expressing . Q97 - GFP expression in wild type leads to many small scattered per . In GAL - Mia40 , Q97 - GFP accumulates to much larger intensities but then suddenly collapses into one single aggregate per . arrow depicts aggregate formation . Bars , 4 µm . C . Quantified signal intensities in of indicated mutants . In top middle graphs , each curve corresponds to a single . Averaged signals ( mean + ) in bottom graph ( N = 20 for WT , N = 17 for GAL - Mia40 ) .

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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 GAL - Mia40 in two biological replicates each . were lysed with NP - 40 before soluble were quantified . Mitochondrial ( mito . ) members of mitochondrial carrier family ( carrier ) are visualized in a volcano plot . P - values were derived from a moderated t - . Mitochondrial ( mito . ) carrier are indicated by yellow red dots , respectively . Rnq1 is indicated in blue .

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

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

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

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

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

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

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

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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 ) . growth is expressed as % change of initial , * * 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 ) . growth is expressed as % change of initial , * * * 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 ) .

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(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 ( 5 x 105 ) were injected into tail vein of CD1 mice . Lung colonization was assayed by HE calculating number of nodules their total normalized per total of lungs . 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 ) .

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(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 of proliferation determined by immunofluorescence Ki67 in A375 xenografts treated as indicated . Bar graphs indicate Ki67 + / ( 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 of apoptosis determined by immunofluorescence with TUNEL in A375 xenografts treated as indicated . Bar graphs indicate TUNEL + / ( n = 3 tumors ) . * * * P < 0 . 001 versus vehicle ( P = 6 . 2 . 7E - 10 ) .

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(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 of vasculature stained by an antibody anti - CD31 in A375 xenografts treated as indicated . Bar graphs indicate quantitative micro vessel density ( MVD ) micro vessel ( MVA ) ( 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 ) .

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(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 of vessel lumen in A375 xenografts treated as indicated . Bar graphs indicate quantitative of lumen diameters in A375 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 ) .

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

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(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 of hypoxia marker pimonidazole in A375 xenografts treated as indicated . Bar graphs show % of hypoxic in A375 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 ) .

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

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

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

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(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 infiltrate was analyzed . Gating strategy for CD45 + graph showing of FACS 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 ) .

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(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 of FACS of infiltrating F4 / 80 + macrophages CD11c + MHCII + dendritic 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 ) .

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(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 indicated genes ( M1 - like 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 ) .

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

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(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 of FACS of infiltrating F4 / 80 + macrophages average of median ( MFI ) of TNFα + or INOS + on F4 / 80 + after ex vivo stimulation with PMA ionomycin in vehicle ( n = 4 tumors ) COMBO ( n = 6 tumors ) , * P < 0 . 05 versus vehicle ( TNFα P = 0 . 037 ; INOS = 0 . 038 ) .

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

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

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(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 ) viability measured as of ZsGreen - A375 co - cultured with 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 ) .

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

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(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 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 housekeeping gene TBP ( n = 3 tumors ) , * P < 0 . 05 versus vehicle ( P = 0 . 043 ) .

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(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 extract from A375 tumors treated as indicate . Vinculin was used as an internal control .

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(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 growth inhibition induced by COMBO treatment in in mice bearing A375 tumors . growth is expressed as % change of initial . Macrophage depletion by clodronate liposome enhanced growth of tumors treated with combination of PLX4720 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of macrophage infiltration determined by F4 / 80 immunofluorescence in A375 xenografts treated as indicated . Bar graphs indicate F4 / 80 + / ( n = 3 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 1 . 53E - 25 ) . ( C ) Representative of collagen deposition determined by collagen I immunofluorescence in A375 xenografts treated as indicated . Bar graphs indicate collagen I + / ( n = 3 tumors ) .

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(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 change in volume ( relative to initial volume ) for A375 xenografts in each treatment group ( vehicle , PLX4720 , bevacizumab COMBO ) from week 1 to week 6 .

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

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(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 ) .

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

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

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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 were treated with 0 . 2 M sorbitol for 30 minutes . Lats1 was immunoprecipitated an in vitro kinase 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 ± . * p < 0 . 05 ( two student ' s t - , n = 3 ) .

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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 localization . HEK293A were treated with 0 . 2 M sorbitol for 1 hr in presence of serum . YAP subcellular localization was determined by immunofluorescence ( red ) . DAPI ( blue ) was used to stain for . Scale bar : 20 μm . of more ( N > C ) or more ( N < C ) YAP signal is determined with randomly chosen fields , each with approximately 100 .

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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 were treated with 0 . 2 M sorbitol for 4 hr . mRNA of CTGF CYR61 were measured by quantitative RT - PCR normalized to GAPDH control . Data are presented as mean ± . n . s means p > 0 . 05 ( two student ' s t - , n = 3 ) .

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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 interaction between YAP TEAD1 . HEK293A were treated with 0 . 2 M sorbitol for 1 hr . Endogenous YAP was immunoprecipitated with YAP antibody , TEAD1 YAP were detected by Western blot . were serum starved for 1 hr as indicated ( - serum ) .

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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 interaction between YAP 14 - 3 - 3 . HEK293A were transiently transfected with Flag - YAP Myc - 14 - 3 - 3 , then treated with 0 . 2 M sorbitol for indicated time points . Myc - 14 - 3 - 3 was immunoprecipitated co - precipitated Flag - YAP was detected .

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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 translocation . HEK293A 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 nuclei . Scale bar : 20 μm . of more ( N > C ) or more ( N < C ) YAP signal is determined with randomly chosen fields .

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

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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 translocation . HEK293A were serum starved for 1 hr followed by 0 . 2 M sorbitol treatment for indicated time points .

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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 were serum starved for 1 hr treated with 0 . 2 M sorbitol for 4 hr . mRNA of CTGF CYR61 were measured by quantitative RT - PCR normalized to GAPDH control . Data are presented as mean ± . * p < 0 . 05 ( two student ' s t - , n = 3 ) .

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

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

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

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 localization . HEK293A 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 absence of serum . of YAP / TAZ subcellular is shown . Scale bar : 20 μm .

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

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 localization . HEK293A 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 absence of serum . lysates from identically treated samples were examined for phosphorylation of p38 substrate MK - 2 JNK substrate c - Jun .

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

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 localization . HEK293A were transiently transfected with CRISPR / Cas9 to knockout NLK . Wild type ( WT ) NLK KO pool were treated with 0 . 2 M sorbitol for 1 hr in absence of serum . YAP / TAZ subcellular localization was determined by immunofluorescence . Scale bar : 20 μm .

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

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

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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 . phos - tag gel showed NLK - WT but not NLK - KN caused a significant mobility shift of YAP .

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

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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 .

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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 immunoprecipitated . An in vitro NLK kinase was performed using recombinant GST - YAP as a substrate . Phosphorylation of YAP Ser 128 was determined by immunoblotting with phospho - YAP ( S128 ) antibody .

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

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

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 two pools of NLK CRISPR / CAS9 transfected were treated with 0 . 2 M sorbitol for indicated time points . Data are presented as mean ± . * p < 0 . 05 ( two student ' s t - , n = 4 ) .

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

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

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 . were serum starved for 16 hrs , treated with 0 . 2 M sorbitol for indicated time points or refreshed with serum containing medium for 1 hr . Refreshing medium served as a control to disrupt YAP 14 - 3 - 3 binding . 14 - 3 - 3 was immunoprecipitated with Myc antibody , associated YAP was detected with Flag antibody . Data are presented as mean ± . * p < 0 . 05 ( two student ' s t - , n = 3 ) .

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

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 translocation of S128A mutant YAP . HEK293A were co - transfected with Myc - NLK Flag - YAP WT or S128A mutant . were serum starved for 6 hr , YAP localization NLK expression were determined by Flag ( green ) Myc ( red ) antibodies , respectively . Scale bar : 20 μm .

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 localization . HEK293A YAP KO 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 ( green ) . Scale bar : 20 μm .

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

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

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

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

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

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

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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 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 ) .

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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 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 ) naked mole - rat ( n = 8 ) . I , Relative 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 .

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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 - Seq datasets . Naked mole - rat BM populations CP1 - 4 were matched to human murine counterparts for GSEA with MSigDB hallmark genesets . Primitive 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 , types were pooled for each species . Shown are pathways related to proliferation metabolism normalized enrichment score ; GeneRatio , ( signature ∩ term ) / ( signature ∩ terms ) ; FDR , false discovery rate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 with 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 types , n = 4 ; mouse , n = 3 , naked mole - rat , n = 3 .

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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.