Figure 3A . Western blot confirming knockdown of Rab11 - FIP1 by shRNAs . Relative intensity by densitometry of Rab11 - FIP1 results are shown at the bottom . 3D organoids were generated from WT and mutant p53 cells expressing non - targeting control ( NT ) or Rab11 - FIP1 shRNAs ( sh1 and sh2 ) . B . Diameter of organoids ( n = 80 - 130 organoids ) . Bar graph represents mean diameter ± SEM . * * * * p < 0 . 0001 WT or mutant p53 non - targeting control vs Rab11 - FIP1 shRNA1 , * * * p = 0 . 0006 WT non - targeting control vs Rab11 - FIP1 shRNA2 , * * * * p < 0 . 0001 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA2 ( One - way Anova ) . * * * * p < 0 . 0001 WT vs mutant p53 non - targeting control ( Unpaired t - test ) . 3D organoids were generated from WT and mutant p53 cells expressing non - targeting control ( NT ) or Rab11 - FIP1 shRNAs ( sh1 and sh2 ) . C . Percent of WT p53 ( top panel ) and mutant ( bottom panel ) organoids exhibiting a solid , intermediate , or stratified morphology ( n > 50 organoids generated from three different cell passages quantified ) . Bar graph represents mean diameter ± SEM . n . s . WT p53 non - targeting control vs Rab11 - FIP1 shRNAs , * p = 0 . 046 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA1 , * * * p = 0 . 0002 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA2 ( One - way Anova ) . 3D organoids were generated from WT and mutant p53 cells expressing non - targeting control ( NT ) or Rab11 - FIP1 shRNAs ( sh1 and sh2 ) . D . Percent of WT p53 and mutant organoids exhibiting a basaloid - like morphology ( n > 50 organoids generated from three different cell passages quantified ) . Bar graph represents mean diameter ± SEM . n . s . WT p53 non - targeting control vs Rab11 - FIP1 shRNAs , * p = 0 . 046 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA1 , * * * p = 0 . 0002 mutant p53 non - targeting control vs Rab11 - FIP1 shRNA2 ( One - way Anova ) . 3D organoids were generated from WT and mutant p53 cells expressing non - targeting control ( NT ) or Rab11 - FIP1 shRNAs ( sh1 and sh2 ) . E . WT p53 organoids stained for H & E ( left ) , Rab11 - FIP1 ( middle ) , and the basal cell marker p63 ( right ) by IHC . 40X . Scale bar = 100 μm . F . Mutant p53 organoids stained for H & E ( left ) , Rab11 - FIP1 ( middle ) , and the basal cell marker p63 ( right ) by IHC . 40X . Scale bar = 100 μm .

O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Western blot confirming knockdown of Rab11-FIP1 by shRNAs. Relative intensity by densitometry of Rab11-FIP1 results are shown at the bottom.3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). B. Diameter of organoids (n= 80-130 organoids). Bar graph represents mean diameter ± SEM. ****p<0.0001 WT or mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0006 WT non-targeting control vs Rab11-FIP1 shRNA2, ****p<0.0001 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova). ****p<0.0001 WT vs mutant p53 non-targeting control (Unpaired t-test).3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). C. Percent of WT p53 (top panel) and mutant (bottom panel) organoids exhibiting a solid, intermediate, or stratified morphology (n>50 organoids generated from three different cell passages quantified). Bar graph represents mean diameter ± SEM. n.s. WT p53 non-targeting control vs Rab11-FIP1 shRNAs, *p=0.046 mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0002 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova).3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). D. Percent of WT p53 and mutant organoids exhibiting a basaloid-like morphology (n>50 organoids generated from three different cell passages quantified). Bar graph represents mean diameter ± SEM. n.s. WT p53 non-targeting control vs Rab11-FIP1 shRNAs, *p=0.046 mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0002 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova).3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). E. WT p53 organoids stained for H&E (left), Rab11-FIP1 (middle), and the basal cell marker p63 (right) by IHC. 40X. Scale bar = 100 μm. F. Mutant p53 organoids stained for H&E (left), Rab11-FIP1 (middle), and the basal cell marker p63 (right) by IHC. 40X. Scale bar = 100 μm.


Figure 4A . EPC2 - hTERT - EGFR - p53R175H cells grown in OTC stained for RCP . Arrow indicates invasive region . ( scale bar = 100 μm ) . B . Transwell Boyden Chamber invasion assay of WT and mutant p53 cells expressing non - targeting control ( NT ) and Rab11 - FIP1 shRNAs ( sh1 and sh2 ) ( n = 9 technical replicates from 3 different passages ) . Error bars represent ± S . E . M . n . s . WT p53 NT vs sh1 , * * * p = 0 . 0005 WT p53 NT vs sh2 , n . s . Mutant p53 NT vs sh1 , * * p = 0 . 0026 Mutant p53 NT vs sh2 , ( One - way Anova ) * p = 0 . 0173 WT p53 NT vs Mutant p53 NT , ( unpaired t - test ) . C - D . WT and mutant p53 cells expressing non - target control and Rab11 - FIP1 shRNAs were grown in OTC . C . H & E staining of OTC with WT p53 ( top ) and mutant p53 ( bottom ) with invasive regions . Scale bar = 50 μm . D . Graph shows relative invasive area from OTC of WT p53 non - target control and Rab11 - FIP1 shRNAs . n = 5 technical replicates in WT p53 NT , n = 6 technical replicates in WT p53 sh1 , n = 6 technical replicates in WT p53 sh2 . Error bars represent ± S . E . M . * * * * p < 0 . 0001 WT p53 NT vs sh1 , n . s . WT p53 NT vs sh2 ( One - way Anova ) .

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Figure 4A. EPC2-hTERT-EGFR-p53R175H cells grown in OTC stained for RCP. Arrow indicates invasive region. (scale bar = 100 μm).B. Transwell Boyden Chamber invasion assay of WT and mutant p53 cells expressing non-targeting control (NT) and Rab11-FIP1 shRNAs (sh1 and sh2) (n=9 technical replicates from 3 different passages). Error bars represent ±S.E.M. n.s. WT p53 NT vs sh1, ***p=0.0005 WT p53 NT vs sh2, n.s. Mutant p53 NT vs sh1,** p=0.0026 Mutant p53 NT vs sh2, (One-way Anova) *p=0.0173 WT p53 NT vs Mutant p53 NT, (unpaired t-test).C-D. WT and mutant p53 cells expressing non-target control and Rab11-FIP1 shRNAs were grown in OTC. C. H&E staining of OTC with WT p53 (top) and mutant p53 (bottom) with invasive regions. Scale bar = 50 μm. D. Graph shows relative invasive area from OTC of WT p53 non-target control and Rab11-FIP1 shRNAs. n=5 technical replicates in WT p53 NT, n=6 technical replicates in WT p53 sh1, n=6 technical replicates in WT p53 sh2. Error bars represent ±S.E.M. **** p<0.0001 WT p53 NT vs sh1, n.s. WT p53 NT vs sh2(One-way Anova).


Figure 5A . Immunofluorescent staining for Rab11 - FIP1 in TE1 and TE2 ESCC cell lines expressing a non - targeting control shRNA or shRNA against Rab11 - FIP1 . Scale bar = 50 μm B . Immunofluorescent staining for f - actin in TE1 and TE2 ESCC cell lines expressing a non - targeting control shRNA or shRNA against Rab11 - FIP1 . Scale bar = 50 μm C . Immunofluorescent staining for E - cadherin , in TE1 and TE2 ESCC cell lines expressing a non - targeting control shRNA or shRNA against Rab11 - FIP1 . Scale bar = 50 μm D . qPCR analysis of epithelial and mesenchymal gene expression in TE1 ( left ) and TE2 ( right ) cells expressing Rab11 - FIP1 shRNA relative to control cells ( dotted line ) . Graph error bars represent mean SEM ( n = 3 technical replicates from three different cell passages ) . n . s . TE2 SNAI2 , * p < 0 . 05 , ( Unpaired t - test ) . E . qPCR analysis of epithelial and mesenchymal gene expression following ZEB1 depletion in TE1 ( left ) and TE2 ( right ) cells expressing Rab11 - FIP1 shRNA relative to control cells ( dotted line ) . Graph error bars represent mean SEM ( n = 3 technical replicates from three different cell passages ) . * * p < 0 . 01 , * p < 0 . 05 , n . s . TE1 CDH1 , VIM . SNAI1 , SNAI2 , TE2 VIM , SNAI1 ( Unpaired t - test ) .

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Figure 5A. Immunofluorescent staining for Rab11-FIP1 in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm B. Immunofluorescent staining for f-actin in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm C. Immunofluorescent staining for E-cadherin, in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm D. qPCR analysis of epithelial and mesenchymal gene expression in TE1 (left) and TE2 (right) cells expressing Rab11-FIP1 shRNA relative to control cells (dotted line). Graph error bars represent mean SEM (n=3 technical replicates from three different cell passages). n.s. TE2 SNAI2, *p<0.05,(Unpaired t-test).E. qPCR analysis of epithelial and mesenchymal gene expression following ZEB1 depletion in TE1 (left) and TE2 (right) cells expressing Rab11-FIP1 shRNA relative to control cells (dotted line). Graph error bars represent mean SEM (n=3 technical replicates from three different cell passages). **p<0.01, * p<0.05, n.s. TE1 CDH1, VIM. SNAI1, SNAI2, TE2 VIM, SNAI1 (Unpaired t-test).


Figure 1Male C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . Serum SUA levelMale C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . body weight gainMale C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . fatMale C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . lean massMale C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . gastrocnemius index . Male C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . Gastrointestinal muscle fiber immunofluorescent laminin stainingMale C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . frequency histogram of fiber cross - sectional area .

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Figure 1Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Serum SUA levelMale C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. body weight gainMale C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. fatMale C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. lean massMale C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. gastrocnemius index.Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Gastrointestinal muscle fiber immunofluorescent laminin stainingMale C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. frequency histogram of fiber cross-sectional area.


Figure 2male C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . The muscle grip strengthmale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . running time in low speedmale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . four - limb handing timemale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . running time in high speed . male C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . serum concentration of RBCmale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . HGB in whole bloodmale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . ex vivo gastrocnemius muscle forcemale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . ex vivo gastrocnemius muscle forcemale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . fatigabilitymale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . glucose consumptionmale C57BL / 6J mice fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . lactate production were tested .

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Figure 2male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. The muscle grip strengthmale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. running time in low speedmale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. four-limb handing timemale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. running time in high speed.male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. serum concentration of RBCmale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. HGB in whole bloodmale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. ex vivo gastrocnemius muscle forcemale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. ex vivo gastrocnemius muscle forcemale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. fatigabilitymale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. glucose consumptionmale C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. lactate production were tested.


Figure 3Male C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . mRNA expression of MyHC I , MyHC IIa , PGC - 1α , myoglobin , TnnT1 MyHC IIb , MyHC IIx and TnnT3 in the gastrocnemius muscle ( n = 5 - 6 ) . Male C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . Immunoblots and quantification of MyHC - I , IIa and IIb protein expression in gastrocnemius ( n = 3 - 4 ) . Male C57BL / 6J mice were fed with chow diet supplemented with 0 , 0 . 5 % , 1 % SUC for 8 weeks . Representative images and quantification of Laminin ( green ) , MyHC - I and IIb immunofluorescent staining ( red ) in gastrocnemius ( n = 3 ) . Scale bar in ( C ) represents 100 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. mRNA expression of MyHC I, MyHC IIa, PGC-1α, myoglobin, TnnT1 MyHC IIb, MyHC IIx and TnnT3 in the gastrocnemius muscle (n=5-6).Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Immunoblots and quantification of MyHC-I, IIa and IIb protein expression in gastrocnemius (n=3-4).Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Representative images and quantification of Laminin (green), MyHC-I and IIb immunofluorescent staining (red) in gastrocnemius (n=3). Scale bar in (C) represents 100 µm.


Figure 4Male C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 weeks . The O2 consumption ( VO2 ) and respiratory exchange ratio ( RER ) Male C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 weeks . Serum concentration of ( E ) NEFA in whole blood . Male C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 weeks . The enzymes activity of ( F ) SDH , ( G ) HK , and ( H ) LDH in gastrocnemius . Male C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 weeks . Immunoblots and quantification of of p - AMPK ， PGC - 1α and myoglobin in gastrocnemius . Male C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 weeks . Quantification of mitochondrial and electron transport chain ( ETC ) related genes expression in gastrocnemius . Male C57BL / 6J mice were fed with chow diet supplemented with 0 and1 % SUC for 6 weeks . OCRs were measured under basal condition in gastrocnemius .

O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. The O2 consumption (VO2) and respiratory exchange ratio (RER)Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Serum concentration of (E) NEFA in whole blood.Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. The enzymes activity of (F) SDH, (G) HK, and (H) LDH in gastrocnemius.Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Immunoblots and quantification of of p-AMPK，PGC-1α and myoglobin in gastrocnemius.Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Quantification of mitochondrial and electron transport chain (ETC) related genes expression in gastrocnemius.Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. OCRs were measured under basal condition in gastrocnemius.


Figure 5C2C12 cells were treated with 0 , 0 . 5 and 2 mM SUC for 48 h . Representative images of MyHC - I and IIb immunofluorescent staining ( green ) in C2C12 cells ( n = 16 ) . C2C12 cells were treated with 0 , 0 . 5 and 2 mM SUC for 48 h . quantification of MyHC - I and IIb immunofluorescent staining ( green ) in C2C12 cells ( n = 16 ) . C2C12 cells were treated with 0 , 0 . 5 and 2 mM SUC for 48 h . The enzymes activity of ( C ) SDH , ( D ) LDHC2C12 cells were treated with 0 , 0 . 5 and 2 mM SUC for 48 h . lactate production in C2C12 cells . C2C12 cells were treated with 0 , 0 . 5 and 2 mM SUC for 48 h . Quantification of mitochondrial DNA contents in C2C12 cells . C2C12 cells were treated with 0 , 0 . 5 and 2 mM SUC for 48 h . Mitochondrial electron microscopy showed the ( H ) mitochondrial density , ( I ) mitochondrial coverage , and ( J ) average mitochondrial area in C2C12 cell . Scale bar in ( A ) represents 50 μm , scale bar in ( G ) represents 0 . 5 μm .

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Figure 5C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Representative images of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=16).C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. quantification of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=16).C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. The enzymes activity of (C) SDH, (D) LDHC2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. lactate production in C2C12 cells.C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Quantification of mitochondrial DNA contents in C2C12 cells.C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Mitochondrial electron microscopy showed the (H) mitochondrial density, (I) mitochondrial coverage, and (J) average mitochondrial area in C2C12 cell. Scale bar in (A) represents 50 μm, scale bar in (G) represents 0.5 μm.


Figure 6SUNCR1 protein expression in the gastrocnemius from sedentary or post - running mice ( n = 4 ) . The mRNA level of SUNCR1 in gastrocnemius and soleus ( n = 7 - 8 ) . [ Ca2 + ] i in C2C12 cells treated with 0 or 2 mM SUC ( n = 18 - 20 ) . NFAT protein expression in nucleus and cytoplasm of gastrocnemius 0 . 5 h or 3 h after i . p . injection of 15 mg / kg succinate in C57BL / 6J mice ( n = 4 ) . SUNCR1 protein expression in C2C12 cells transfected with vector or siSUNCR1 ( n = 3 ) . [ Ca2 + ] i , in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC ( n = 5 - 6 ) . enzymes activity ( n = 9 - 10 ) of ( J ) HK , ( K ) LDH , and ( L ) SDH in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC ( n = 5 - 6 ) . Representative images and quantification of MyHC - I and IIb immunofluorescent staining ( green ) in C2C12 cells ( n = 3 ) . Scale bar in ( M ) represents 50 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6SUNCR1 protein expression in the gastrocnemius from sedentary or post-running mice (n=4).The mRNA level of SUNCR1 in gastrocnemius and soleus (n=7-8).[Ca2+]i in C2C12 cells treated with 0 or 2 mM SUC (n=18-20).NFAT protein expression in nucleus and cytoplasm of gastrocnemius 0.5 h or 3 h after i. p. injection of 15 mg/kg succinate in C57BL/6J mice (n=4).SUNCR1 protein expression in C2C12 cells transfected with vector or siSUNCR1 (n=3).[Ca2+]i, in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC (n=5-6).enzymes activity (n=9-10)of (J) HK, (K) LDH, and (L) SDH in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC (n=5-6).Representative images and quantification of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=3). Scale bar in (M) represents 50 μm.


Figure 7Male C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . The O2 consumption ( VO2 ) , RERMale C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . muscle grip strengthMale C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . four - limb handing timeMale C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . low speed running time . Male C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . The enzymes activity of ( H ) HK , ( I ) LDH , and ( J ) SDH in gastrocnemius . Male C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . Immunoblots and quantification of MyHC - I , IIb , NFAT , and PGC - 1α protein in gastrocnemius . Male C57BL / 6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . Representative images and quantification of Laminin ( green ) , or MyHC - I and IIb ( red ) immunofluorescent staining in gastrocnemius muscle ( n = 3 ) . Scale bar in ( M ) represents 100 µm .

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Figure 7Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The O2 consumption (VO2), RERMale C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. muscle grip strengthMale C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. four-limb handing timeMale C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. low speed running time.Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The enzymes activity of (H) HK, (I) LDH, and (J) SDH in gastrocnemius.Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Immunoblots and quantification of MyHC-I, IIb, NFAT, and PGC-1α protein in gastrocnemius.Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Representative images and quantification of Laminin (green), or MyHC-I and IIb (red) immunofluorescent staining in gastrocnemius muscle (n=3). Scale bar in (M) represents 100 µm.


Figure 8Male C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age . After two weeks of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . SUNCR1 protein expression in gastrocnemius from mice transfected with shSUNCR1 lentivirus or LV - shScrambled ( n = 3 ) . Male C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age . After two weeks of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . The running time in low speedMale C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age . After two weeks of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . four - limb handing timeMale C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age . After two weeks of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . muscle grip strength of both control and gastrocnemius - specific SUNCR1 knockdown mice . Male C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age . After two weeks of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . The enzymes activity of ( F ) HK , ( G ) LDH , ( H ) SDH in gastrocnemius . Male C57BL / 6J mice were injected with LV - shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age . After two weeks of recovery , mice were fed with chow diet supplemented with 0 or 1 % SUC for 6 weeks . Immunoblots and quantification of MyHC - I , IIb , NFAT , and PGC - 1α protein in gastrocnemius ( n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. SUNCR1 protein expression in gastrocnemius from mice transfected with shSUNCR1 lentivirus or LV-shScrambled (n=3).Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The running time in low speedMale C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. four-limb handing timeMale C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. muscle grip strength of both control and gastrocnemius-specific SUNCR1 knockdown mice.Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The enzymes activity of (F) HK, (G) LDH, (H) SDH in gastrocnemius.Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Immunoblots and quantification of MyHC-I, IIb, NFAT, and PGC-1α protein in gastrocnemius (n=3).


Figure 1A , B 1 × 105 B16F10 melanoma cells were intravenously injected into C57BL / 6 wild - type ( WT ) or Asm - deficient ( Asm − / − ) mice , and the number of lung metastases was determined after 14 days . The photographs ( A ) show two representative results . The graph ( B ) shows the mean ± SD number of pulmonary metastases in 15 WT and 14 Asm - deficient animals . C , D B16F10 melanoma grow as fast or slightly faster in Asm - deficient mice than in wild - type mice after subcutaneous injection at the flank ( C ) or transcutaneous direct intrapulmonary injection ( D ) , excluding a general growth defect of the tumor in Asm - deficient mice . The size of tumors was determined 14 days after local injection at the flank or 10 days after injection into the lung . E The number of lung metastases 25 days after intravenous injection does not differ from the number observed after 14 days in ( B ) . F 5 × 108platelets isolated from wild - type ( WT ) or Asm - deficient ( Asm − / − ) mice were injected intravenously into Asm - deficient or wild - type mice , respectively . After 120 min 1 × 105 , B16F10tumor cells were intravenously injected . The number of lungmetastases was determined after 14 days . Displayed are the mean ± SD of the number of pulmonary metastases , n = 3 in WT platelets in WT mice , n = 5 in WT platelets in Asm − / − mice , and n = 4 in Asm − / − platelets in Asm − / − mice .

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Figure 1A, B 1 × 105 B16F10 melanoma cells were intravenously injected into C57BL/6 wild-type (WT) or Asm-deficient (Asm−/−) mice, and the number of lung metastases was determined after 14 days. The photographs (A) show two representative results. The graph (B) shows the mean ± SD number of pulmonary metastases in 15 WT and 14 Asm-deficient animals.C, D B16F10 melanoma grow as fast or slightly faster in Asm-deficient mice than in wild-type mice after subcutaneous injection at the flank (C) or transcutaneous direct intrapulmonary injection (D), excluding a general growth defect of the tumor in Asm-deficient mice. The size of tumors was determined 14 days after local injection at the flank or 10 days after injection into the lung.E The number of lung metastases 25 days after intravenous injection does not differ from the number observed after 14 days in (B).F 5 × 108platelets isolated from wild-type (WT) or Asm-deficient (Asm−/−) mice were injected intravenously into Asm-deficient or wild-type mice, respectively. After 120 min 1 × 105, B16F10tumor cells were intravenously injected. The number of lungmetastases was determined after 14 days. Displayed are the mean ± SD of the number of pulmonary metastases, n = 3 in WT platelets in WT mice, n = 5 in WT platelets in Asm−/−mice, and n = 4 in Asm−/−platelets in Asm−/−mice.


Figure 2A Platelets were isolated from wild - type ( WT ) or Asm - deficient ( Asm − / − ) mice by density - gradient centrifugation , and 1 × 107 platelets were incubated with 1 × 105 B16F10 cells . The samples were lysed , and Asm activity was determined . In unstimulated samples ( time point of co - incubation 0 s ) , tumor cells and platelets were admixed after lysis . B The formation of ceramide was determined by a diacylglycerol ( DAG ) kinase assay ( upper panel ) and mass spectrometry ( lower panel ) . In unstimulated samples ( time point of co - incubation 0 s ) , tumor cells and platelets were admixed after lysis . C , D For determination of the secretion of Asm by platelets into the supernatants ( C ) , tumor cells and wild - type ( WT ) or Asm - deficient ( Asm − / − ) platelets were co - incubated for 20 s , the samples were pelleted , the supernatants were removed and acidified , and the Asm activity was measured in the presence or absence of Zn2 + . For measurement of surface Asm and ceramide , samples were co - incubated as indicated ( C , D ) and pelleted ; the supernatants were discarded , incubated with anti - Asm antibodies , washed , and lysed ; and Asm immunocomplexes were immobilized and subjected to immunocomplex enzyme assays . Surface ceramide was measured by incubation of intact cells with DAG kinase in the presence of [ 32P ] γATP followed by extraction and measurement of [ 32P ] - ceramide . Omission of one cell type indicated by " − " here and thereafter . E Treatment of B16F10tumor cells for 2 min with 1 U / ml ASM or 10 μM C16ceramide restores in vivo metastasis in Asm - deficient mice . After treatment , the cells were injected intravenously into Asm - deficient ( Asm − / − ) mice . Controls were left untreated prior to injection . The number of metastases was determined 14 days after tumor cell injection .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Platelets were isolated from wild-type (WT) or Asm-deficient (Asm−/−) mice by density-gradient centrifugation, and 1 × 107 platelets were incubated with 1 × 105 B16F10 cells. The samples were lysed, and Asm activity was determined. In unstimulated samples (time point of co-incubation 0 s), tumor cells and platelets were admixed after lysis.B The formation of ceramide was determined by a diacylglycerol (DAG) kinase assay (upper panel) and mass spectrometry (lower panel). In unstimulated samples (time point of co-incubation 0 s), tumor cells and platelets were admixed after lysis.C, D For determination of the secretion of Asm by platelets into the supernatants (C), tumor cells and wild-type (WT) or Asm-deficient (Asm−/−) platelets were co-incubated for 20 s, the samples were pelleted, the supernatants were removed and acidified, and the Asm activity was measured in the presence or absence of Zn2+. For measurement of surface Asm and ceramide, samples were co-incubated as indicated (C, D) and pelleted; the supernatants were discarded, incubated with anti-Asm antibodies, washed, and lysed; and Asm immunocomplexes were immobilized and subjected to immunocomplex enzyme assays. Surface ceramide was measured by incubation of intact cells with DAG kinase in the presence of [32P]γATP followed by extraction and measurement of [32P]-ceramide. Omission of one cell type indicated by "−" here and thereafter.E Treatment of B16F10tumor cells for 2 min with 1 U/ml ASM or 10 μM C16ceramide restores in vivo metastasis in Asm-deficient mice. After treatment, the cells were injected intravenously into Asm-deficient (Asm−/−) mice. Controls were left untreated prior to injection. The number of metastases was determined 14 days after tumor cell injection.


Figure 3Incubation of human melanoma ( HM ) cells with human platelets results in the release of Zn2 + - dependent ASM into the supernatant , Zn2 + - dependent activity of ASM on cell surfaces , and the formation of ceramide . The assay buffer contained 100 μM Zn2 + . Addition of human or mouse recombinant ASM to human melanoma or B16F10 cells , respectively , results in binding of ASM to the tumorcell surfaces as determined by flow cytometry . Cytochalasin B was added to control for internalization of added ASM . Suppression of Asm in B16F10 tumor cells using siRNA technology reduces Asm activity in B16F10 cells ( right panel ) , but does not alter release or surface activity of the Asm after co - incubation with wild - type platelets ( left panels ) .

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Figure 3Incubation of human melanoma (HM) cells with human platelets results in the release of Zn2+-dependent ASM into the supernatant, Zn2+-dependent activity of ASM on cell surfaces, and the formation of ceramide. The assay buffer contained 100 μM Zn2+.Addition of human or mouse recombinant ASM to human melanoma or B16F10 cells, respectively, results in binding of ASM to the tumorcell surfaces as determined by flow cytometry. Cytochalasin B was added to control for internalization of added ASM.Suppression of Asm in B16F10 tumor cells using siRNA technology reduces Asm activity in B16F10 cells (right panel), but does not alter release or surface activity of the Asm after co-incubation with wild-type platelets (left panels).


Figure 4Platelets were isolated from wild - type ( WT ) or Asm - deficient ( Asm − / − ) mice by density - gradient centrifugation , and 1 × 107platelets were incubated with 1 × 105B16F10 cells or left untreated . Co - incubation of washed platelets with B16F10 melanoma cells resulted in upregulation of GPIIb / IIIa and CD62P on both wild - type and Asm - deficient platelets in FACS analyses . Platelets were gated in FCS / SSC . Aggregation properties of platelets after co - incubation with collagen or ADP are independent of Asm as determined by aggregometry measurements . Incubation of tumor cells with platelets resulted in marked change of platelet shape and degranulation as determined by FACS analysis using forward versus side scatter analysis indicating global activation of platelets . 4 × 104B16F10tumor cells were incubated with 2 × 107 wild - type ( WT ) , Asm - deficient ( Asm − / − ) platelets ( Plts ) or a 45 : 55 of WT : Asm − / − platelet mixture in the presence or absence of 50 ng / ml PGE1 . Controls were stimulated with Mn2 + . Tumor cells were then incubated for 60 s on fibronectin - coated cover slips , washed , and fixed , and adhesion of the tumor cells was determined . The graph displays the mean ± SD of tumor cells adhering to fibronectin - coated cover slips , n = 7 for WT - platelets , n = 9 for Asm - deficient - platelets , n = 4 for 45 : 55 WT : Asm − / − platelets , and all others n = 3 . Statistical significance was determined by analysis of variance ( ANOVA ) followed by Tukey ' s multiple comparisons test . P - values are indicated .

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Figure 4Platelets were isolated from wild-type (WT) or Asm-deficient (Asm−/−) mice by density-gradient centrifugation, and 1 × 107platelets were incubated with 1 × 105B16F10 cells or left untreated. Co-incubation of washed platelets with B16F10 melanoma cells resulted in upregulation of GPIIb/IIIa and CD62P on both wild-type and Asm-deficient platelets in FACS analyses. Platelets were gated in FCS/SSC.Aggregation properties of platelets after co-incubation with collagen or ADP are independent of Asm as determined by aggregometry measurements.Incubation of tumor cells with platelets resulted in marked change of platelet shape and degranulation as determined by FACS analysis using forward versus side scatter analysis indicating global activation of platelets.4 × 104B16F10tumor cells were incubated with 2 × 107 wild-type (WT), Asm-deficient (Asm−/−) platelets (Plts) or a 45:55 of WT:Asm−/−platelet mixture in the presence or absence of 50 ng/ml PGE1. Controls were stimulated with Mn2+. Tumor cells were then incubated for 60 s on fibronectin-coated cover slips, washed, and fixed, and adhesion of the tumor cells was determined. The graph displays the mean ± SD of tumor cells adhering to fibronectin-coated cover slips, n = 7 for WT-platelets, n = 9 for Asm-deficient-platelets, n = 4 for 45:55 WT:Asm−/−platelets, and all others n = 3. Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. P-values are indicated.


Figure 5A - C 1 × 105 B16F10 cells were incubated with ( A ) 1 U / ml purified acid sphingomyelinase ( ASM ) or ( B ) 5 × 107 wild - type ( WT ) , Asm - deficient ( Asm − / − ) platelets ( Plts ) , or a mixture of 45 : 55 wild - type : Asm - deficient platelets . Cells were fixed with 2 % paraformaldehyde for 15 min and stained with fluorescein isothiocyanate ( FITC ) - coupled anti - α5 integrin , Cy3 - labeled anti - β1 integrin , and Cy5 - labeled anti - ceramide antibodies . Samples were analyzed by confocal microscopy . Shown are representative examples from four independent experiments ( A , B ) or the quantitative analysis of cells positive for ceramide / β1 integrin clusters from at least 100 cells / sample ( C ) . Given is the mean ± SD , n = 4 , ANOVA followed by Tukey ' s multiple comparisons test . P - values are indicated . D ASM - induced B16F10 tumorcell adhesion to fibronectin - coated cover slips is abrogated by the inhibition of integrins with RGD peptides . Shown is the mean ± SD of the number of cells adherent to the cover slip . Statistical significance was determined by ANOVA followed by Tukey ' s multiple comparisons test . P - values are indicated . E Intravenous injection of CFSE - labeled B16F10 cells into wild - type mice results in formation of ceramide - enriched domains that contain β1 integrin clusters on tumor cells in vivo , while injection of tumor cells into Asm - deficient mice does not result in ceramide and integrin clustering . Please note that the cell in the lower panel still binds a platelet . Shown are representative results from four independent experiments each .

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Figure 5A-C 1 × 105 B16F10 cells were incubated with (A) 1 U/ml purified acid sphingomyelinase (ASM) or (B) 5 × 107 wild-type (WT), Asm-deficient (Asm−/−) platelets (Plts), or a mixture of 45:55 wild-type:Asm-deficient platelets. Cells were fixed with 2% paraformaldehyde for 15 min and stained with fluorescein isothiocyanate (FITC)-coupled anti-α5 integrin, Cy3-labeled anti-β1 integrin, and Cy5-labeled anti-ceramide antibodies. Samples were analyzed by confocal microscopy. Shown are representative examples from four independent experiments (A, B) or the quantitative analysis of cells positive for ceramide/β1 integrin clusters from at least 100 cells/sample (C). Given is the mean ± SD, n = 4, ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.D ASM-induced B16F10 tumorcell adhesion to fibronectin-coated cover slips is abrogated by the inhibition of integrins with RGD peptides. Shown is the mean ± SD of the number of cells adherent to the cover slip. Statistical significance was determined by ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.E Intravenous injection of CFSE-labeled B16F10 cells into wild-type mice results in formation of ceramide-enriched domains that contain β1 integrin clusters on tumor cells in vivo, while injection of tumor cells into Asm-deficient mice does not result in ceramide and integrin clustering. Please note that the cell in the lower panel still binds a platelet. Shown are representative results from four independent experiments each.


Figure 6Human melanoma cells were stimulated for 10 min with 1 U / ml purified ASM or 10 μM C16 ceramide ( C16 - Cer ) in the presence or absence of 500 ng ceramidase ( CDase ) or 1 μg / ml neutralizing anti - ceramide antibodies ( anti - Cer ) , and lysed and active β1 integrin was immunoprecipitated using the HUTS - 4 antibody . Octylglucopyranoside ( OGP , final concentration 0 . 01 % ) served to resuspend C16 ceramide . Internalization was excluded by addition of cytochalasin B ( CTB ) as indicated . Samples were separated by SDS - PAGE and blotted with an activation - independent anti - β1 Integrin antibody to detect the amount of immunoprecipitated , that is active β1 integrin . Control immunoprecipitates ( control Ipt ) were performed with an irrelevant isotype control antibody . Activation of β1 integrin in human melanoma cells by incubation with Asm or C16 ceramide was confirmed by FACS analysis upon staining with FITC - labeled HUTS - 4 antibodies . Incubation of human melanoma with human platelets or ASM for 5 min triggers co - clustering of ceramide and activated β1 integrin on the surface of the tumor cells . Cells were stained with FITC - coupled anti - active β1 integrin ( HUTS - 4 ) antibodies and Cy3 - coupled anti - ceramide antibodies . Shown are representative stainings from each four independent experiments .

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Figure 6Human melanoma cells were stimulated for 10 min with 1 U/ml purified ASM or 10 μM C16 ceramide (C16-Cer) in the presence or absence of 500 ng ceramidase (CDase) or 1 μg/ml neutralizing anti-ceramide antibodies (anti-Cer), and lysed and active β1 integrin was immunoprecipitated using the HUTS-4 antibody. Octylglucopyranoside (OGP, final concentration 0.01%) served to resuspend C16 ceramide. Internalization was excluded by addition of cytochalasin B (CTB) as indicated. Samples were separated by SDS-PAGE and blotted with an activation-independent anti-β1 Integrin antibody to detect the amount of immunoprecipitated, that is active β1 integrin. Control immunoprecipitates (control Ipt) were performed with an irrelevant isotype control antibody. Activation of β1 integrin in human melanoma cells by incubation with Asm or C16 ceramide was confirmed by FACS analysis upon staining with FITC-labeled HUTS-4 antibodies.Incubation of human melanoma with human platelets or ASM for 5 min triggers co-clustering of ceramide and activated β1 integrin on the surface of the tumor cells. Cells were stained with FITC-coupled anti-active β1 integrin (HUTS-4) antibodies and Cy3-coupled anti-ceramide antibodies. Shown are representative stainings from each four independent experiments.


Figure 7B16F10tumor cells or B16F10 transfected with siRNA targeting Asm expression or with scrambled control siRNA were labeled with [ 3H ] thymidine , washed , and treated ( + ) for 10 min with 1 U / ml ASM , 10 μM C16ceramide , 10 μM S1P , 10 μM PDMP , 20 μM sphingosine kinase inhibitor SKI II , and 10 μM myriocin , or left untreated ( − ) . The cells were then injected intravenously into wild - type or Asm - deficient mice as indicated . If indicated , 10 μg of arginine - glycine - aspartic acid ( RGD ) peptide or 10 μg / ml neutralizing anti - β1 integrin antibodies were added to B16F10tumor cells together with ASM for 15 min prior injection . Mice were sacrificed 30 min after tumorcell injection , the lungs carefully flushed via the right heart , and the radioactivity in the lung as a measurement for adherent tumor cells was determined . Shown is the mean ± SD of the counts per minute ( cpm ) in the lung lysates from three independent experiments . Statistical significance was determined by analysis of variance ( ANOVA ) followed by Tukey ' s test for multiple comparison to determine P - values .

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Figure 7B16F10tumor cells or B16F10 transfected with siRNA targeting Asm expression or with scrambled control siRNA were labeled with [3H]thymidine, washed, and treated (+) for 10 min with 1 U/ml ASM, 10 μM C16ceramide, 10 μM S1P, 10 μM PDMP, 20 μM sphingosine kinase inhibitor SKI II, and 10 μM myriocin, or left untreated (−). The cells were then injected intravenously into wild-type or Asm-deficient mice as indicated. If indicated, 10 μg of arginine-glycine-aspartic acid (RGD) peptide or 10 μg/ml neutralizing anti-β1 integrin antibodies were added to B16F10tumor cells together with ASM for 15 min prior injection. Mice were sacrificed 30 min after tumorcell injection, the lungs carefully flushed via the right heart, and the radioactivity in the lung as a measurement for adherent tumor cells was determined. Shown is the mean ± SD of the counts per minute (cpm) in the lung lysates from three independent experiments. Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey's test for multiple comparison to determine P-values.


Figure 8A Amitriptyline ( 2 mg / kg , Ami ) was intraperitoneally injected into C57BL / 6mice for five times every 12 h . Sixty minutes after the last injection , 1 × 105B16F10tumor cells were intravenously injected . Control experiments confirmed that amitriptyline inhibited Asm activity in the blood by approximately 85 % . Asm heterozygous mice were injected with 1 × 105B16F10tumor cells . The number of lungmetastases was determined after 14 days . Shown is the mean ± SD from six mice each , ANOVA followed by Tukey ' s test for multiple comparison . P - values are indicated . B , C MT / ret cells ( 105 ) suspended in 200 μl Matrigel / PBS ( 1 : 1 ) were injected s . c . in the left and right flanks of 8 - week - old Asm - deficient mice or wild - type mice . Mice were sacrificed at day 20 after injection , and spleens were inspected for the presence of metastases . The representative photographs ( B ) show the results from one of four experiments , and the quantitative analysis is displayed in ( C ) . B , C MT / ret cells ( 105 ) suspended in 200 μl Matrigel / PBS ( 1 : 1 ) were injected s . c . in the left and right flanks of 8 - week - old Asm - deficient mice or wild - type mice . Mice were sacrificed at day 20 after injection , and spleens were inspected for the presence of metastases . The representative photographs ( B ) show the results from one of four experiments , and the quantitative analysis is displayed in ( C ) . D Platelets were isolated from wild - type or Asm - deficient mice by density - gradient centrifugation , and 1 × 107 platelets were incubated with 1 × 105 MT / ret melanoma cells . The samples were lysed , and Asm activity in cell lysates was determined . In unstimulated samples ( time point 0 of co - incubation ) , tumorcell and platelet lysates were admixed after lysis . E To measure secretion of Asm by platelets , tumor cells and platelets were co - incubated for 30 s , the samples were pelleted , the supernatants were acidified , and the Asm activity was measured . All Asm activity measurements were performed in the presence of 100 μM Zn2 + . F Local tumor growth in wild - type or Asm - deficient mice at the flank was measured at day 20 .

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Figure 8A Amitriptyline (2 mg/kg, Ami) was intraperitoneally injected into C57BL/6mice for five times every 12 h. Sixty minutes after the last injection, 1 × 105B16F10tumor cells were intravenously injected. Control experiments confirmed that amitriptyline inhibited Asm activity in the blood by approximately 85%. Asm heterozygous mice were injected with 1 × 105B16F10tumor cells. The number of lungmetastases was determined after 14 days. Shown is the mean ± SD from six mice each, ANOVA followed by Tukey's test for multiple comparison. P-values are indicated.B, C MT/ret cells (105) suspended in 200 μl Matrigel/PBS (1:1) were injected s.c. in the left and right flanks of 8-week-old Asm-deficient mice or wild-type mice. Mice were sacrificed at day 20 after injection, and spleens were inspected for the presence of metastases. The representative photographs (B) show the results from one of four experiments, and the quantitative analysis is displayed in (C).B, C MT/ret cells (105) suspended in 200 μl Matrigel/PBS (1:1) were injected s.c. in the left and right flanks of 8-week-old Asm-deficient mice or wild-type mice. Mice were sacrificed at day 20 after injection, and spleens were inspected for the presence of metastases. The representative photographs (B) show the results from one of four experiments, and the quantitative analysis is displayed in (C).D Platelets were isolated from wild-type or Asm-deficient mice by density-gradient centrifugation, and 1 × 107 platelets were incubated with 1 × 105 MT/ret melanoma cells. The samples were lysed, and Asm activity in cell lysates was determined. In unstimulated samples (time point 0 of co-incubation), tumorcell and platelet lysates were admixed after lysis.E To measure secretion of Asm by platelets, tumor cells and platelets were co-incubated for 30 s, the samples were pelleted, the supernatants were acidified, and the Asm activity was measured. All Asm activity measurements were performed in the presence of 100 μM Zn2+.F Local tumor growth in wild-type or Asm-deficient mice at the flank was measured at day 20.


Figure 1A ) Single dose AR - 42 plasma and tissue pharmacokinetic study . Tumor - free CD2F1 mice were administered a single dose of 10 , 20 or 50 mg / kg AR - 42 ( n = 3 ) and plasma ( dashed ) and gastrocnemius ( solid ) tissue were analyzed for AR - 42 content at different times using LC - MS / MS analyses according to the Materials and Methods ( Mean ± SD ) . B ) AR - 42 dose - response . Starting six days after C - 26 cell injection , animals received vehicle or AR - 42 orally at 1 , 3 , 10 or 20 mg / kg daily or 50 mg / kg every other day for 13 days . Individual animal terminal ( Day 18 post - injection ) body weights corrected for tumor mass according to the Materials and Methods are compared to tumor - free controls . Groups : 1mg / kg ( n = 8 ) , 3mg / kg ( n = 8 ) , 10mg / kg ( n = 13 ) , 20mg / kg ( n = 6 ) , 50mg / kg ( n = 5 ) . Dashed reference line ( 100 % ) represents the mean tumor - free control value , solid line represents non - linear fit of dose - response data . Study 1 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 5 ) , AR - 42 ( 10 mg / kg ; n = 5 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 5 ) or Vehicle ( n = 5 ) and tumor - free mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . C ) : Terminal ( Day 18 post - injection ) body weights corrected for tumor mass according to the Materials and Methods compared to tumor - free controls ( mean ± SD ) . Study 1 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 5 ) , AR - 42 ( 10 mg / kg ; n = 5 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 5 ) or Vehicle ( n = 5 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . D ) Terminal tumor volumes ( mean ± SD ) . Study 1 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 5 ) , AR - 42 ( 10 mg / kg ; n = 5 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 5 ) or Vehicle ( n = 5 ) and tumor - free mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . E ) Terminal gastrocnemius ( mean ± SD ) and F ) quadriceps muscle mass ( mean ± SD ) compared to tumor - free controls .

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Figure 1A) Single dose AR-42 plasma and tissue pharmacokinetic study. Tumor-free CD2F1 mice were administered a single dose of 10, 20 or 50 mg/kg AR-42 (n=3) and plasma (dashed) and gastrocnemius (solid) tissue were analyzed for AR-42 content at different times using LC-MS/MS analyses according to the Materials and Methods (Mean±SD).B) AR-42 dose-response. Starting six days after C-26 cell injection, animals received vehicle or AR-42 orally at 1, 3, 10 or 20 mg/kg daily or 50 mg/kg every other day for 13 days. Individual animal terminal (Day 18 post-injection) body weights corrected for tumor mass according to the Materials and Methods are compared to tumor-free controls. Groups: 1mg/kg (n=8), 3mg/kg (n=8), 10mg/kg (n=13), 20mg/kg (n=6), 50mg/kg (n=5). Dashed reference line (100%) represents the mean tumor-free control value, solid line represents non-linear fit of dose-response data.Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) and tumor-free mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. C): Terminal (Day 18 post-injection) body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. D) Terminal tumor volumes (mean±SD).Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) and tumor-free mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. E) Terminal gastrocnemius (mean±SD) and F) quadriceps muscle mass (mean±SD) compared to tumor-free controls.


Figure 2Study 2 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 10 ) , AR - 42 ( 10 mg / kg ; n = 9 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 9 ) or Vehicle ( n = 7 ) and tumor - free male mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 12 days starting 6 days post - injection of C - 26 cells . A ) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor - free controls ( mean ± SD ) . Study 2 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 10 ) , AR - 42 ( 10 mg / kg ; n = 9 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 9 ) or Vehicle ( n = 7 ) and tumor - free male mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 12 days starting 6 days post - injection of C - 26 cells . B ) Terminal gastrocnemius ( mean ± SD ) and C ) quadriceps muscle mass ( mean ± SD ) compared to tumor - free controls . Study 2 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 10 ) , AR - 42 ( 10 mg / kg ; n = 9 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 9 ) or Vehicle ( n = 7 ) and tumor - free male mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 12 days starting 6 days post - injection of C - 26 cells . D ) grip strength ( mean ± SD ) compared to tumor - free controls . Statistics : V , G , A indicate significant differences versus tumor - bearing vehicle - treated , tumor - bearing GTx - 024 - treated and tumor - bearing AR - 42 - treated groups , respectively . p values provided in Appendix Table S2A - D , one - way ANOVA followed by Tukey ' s multiple comparison test . E ) Survival analyses of Study 2 animals performed as outlined in the Materials and Methods . Statistics : V , G indicate significant differences versus tumor - bearing vehicle - treated and tumor - bearing GTx - 024 - treated groups , respectively . p = 0 . 0005 for Combo vs . V , p = 0 . 0064 for Combo vs . G , Logrank ( Mantel - Cox ) test using Bonferroni corrected threshold for multiple comparisons .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Study 2, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=10), AR-42 (10 mg/kg; n=9), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=9) or Vehicle (n=7) and tumor-free male mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 12 days starting 6 days post-injection of C-26 cells. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).Study 2, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=10), AR-42 (10 mg/kg; n=9), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=9) or Vehicle (n=7) and tumor-free male mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 12 days starting 6 days post-injection of C-26 cells. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.Study 2, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=10), AR-42 (10 mg/kg; n=9), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=9) or Vehicle (n=7) and tumor-free male mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 12 days starting 6 days post-injection of C-26 cells. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, G, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing GTx-024-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S2A-D, one-way ANOVA followed by Tukey's multiple comparison test.E) Survival analyses of Study 2 animals performed as outlined in the Materials and Methods. Statistics: V, G indicate significant differences versus tumor-bearing vehicle-treated and tumor-bearing GTx-024-treated groups, respectively. p=0.0005 for Combo vs. V, p=0.0064 for Combo vs. G, Logrank (Mantel-Cox) test using Bonferroni corrected threshold for multiple comparisons.


Figure 3Study 3 , tumor - bearing female mice receiving GTx - 024 ( 15 mg / kg ; n = 12 ) , AR - 42 ( 10 mg / kg ; n = 12 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 12 ) or Vehicle ( n = 12 ) and tumor - free mice receiving Vehicle ( n = - 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . A ) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor - free controls ( mean ± SD ) . Study 3 , tumor - bearing female mice receiving GTx - 024 ( 15 mg / kg ; n = 12 ) , AR - 42 ( 10 mg / kg ; n = 12 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 12 ) or Vehicle ( n = 12 ) and tumor - free mice receiving Vehicle ( n = - 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . B ) Terminal gastrocnemius ( mean ± SD ) and C ) quadriceps muscle mass ( mean ± SD ) compared to tumor - free controls . Study 3 , tumor - bearing female mice receiving GTx - 024 ( 15 mg / kg ; n = 12 ) , AR - 42 ( 10 mg / kg ; n = 12 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 12 ) or Vehicle ( n = 12 ) and tumor - free mice receiving Vehicle ( n = - 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . D ) grip strength ( mean ± SD ) compared to tumor - free controls . Statistics : V , G , A indicate significant differences versus tumor - bearing vehicle - treated , tumor - bearing GTx - 024 - treated and tumor - bearing AR - 42 - treated groups , respectively . p values provided in Appendix Table S3A - D , one - way ANOVA followed by Tukey ' s multiple comparison test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD) .Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, G, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing GTx-024-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S3A-D, one-way ANOVA followed by Tukey's multiple comparison test.


Figure 4Study 4 , tumor - bearing male mice receiving AR - 42 ( 10 mg / kg , oral gavage ; n = 9 ) , TFM - 4AS - 1 ( 10 mg / kg , subcutaneous ; n = 10 ) , Combination of AR - 42 and DHT ( 10 mg / kg oral gavage and 3 mg / kg subcutaneous , respectively ; n = 10 ) , Combination of AR - 42 and TFM - 4AS - 1 ( 10 mg / kg , both , n = 9 ) or Vehicle ( n = 6 ) and tumor free mice receiving Vehicle ( n = 6 ) were treated daily for 12 days starting 6 days after cell injection . A ) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor - free controls ( mean ± SD ) . Study 4 , tumor - bearing male mice receiving AR - 42 ( 10 mg / kg , oral gavage ; n = 9 ) , TFM - 4AS - 1 ( 10 mg / kg , subcutaneous ; n = 10 ) , Combination of AR - 42 and DHT ( 10 mg / kg oral gavage and 3 mg / kg subcutaneous , respectively ; n = 10 ) , Combination of AR - 42 and TFM - 4AS - 1 ( 10 mg / kg , both , n = 9 ) or Vehicle ( n = 6 ) and tumor free mice receiving Vehicle ( n = 6 ) were treated daily for 12 days starting 6 days after cell injection . B ) Terminal gastrocnemius ( mean ± SD ) and C ) quadriceps muscle mass ( mean ± SD ) compared to tumor - free controls . Study 4 , tumor - bearing male mice receiving AR - 42 ( 10 mg / kg , oral gavage ; n = 9 ) , TFM - 4AS - 1 ( 10 mg / kg , subcutaneous ; n = 10 ) , Combination of AR - 42 and DHT ( 10 mg / kg oral gavage and 3 mg / kg subcutaneous , respectively ; n = 10 ) , Combination of AR - 42 and TFM - 4AS - 1 ( 10 mg / kg , both , n = 9 ) or Vehicle ( n = 6 ) and tumor free mice receiving Vehicle ( n = 6 ) were treated daily for 12 days starting 6 days after cell injection . D ) grip strength ( mean ± SD ) compared to tumor - free controls . Statistics : V , T , A indicate significant differences versus tumor - bearing vehicle - treated , tumor - bearing TFM - 4AS - 1 - treated and tumor - bearing AR - 42 - treated groups , respectively . p values provided in Appendix Table S4A - D , Tukey ' s multiple comparison test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, T, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing TFM-4AS-1-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S4A-D, Tukey's multiple comparison test.


Figure 5A ) Gene expression of multiple cachexia - associated markers in gastrocnemius muscles of individual animals from Study 1 ( n = 5 for tumor - bearing groups ; n = 6 for tumor - free groups ) . Expression was determined by qRT - PCR and presented as described in the Materials and Methods ( Geometric Mean ± Geometric STD ) . B ) Androgen receptor ( AR ) mRNA expression in gastrocnemius muscles from Study 1 . Statistics : p values provided in Appendix Table S5A - B . Dunnett ' s multiple comparison test . CEBPδ , n = 4 , insufficient sample to analyze all tumor - bearing GTx - 024 - treated animals .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) Gene expression of multiple cachexia-associated markers in gastrocnemius muscles of individual animals from Study 1 (n=5 for tumor-bearing groups; n=6 for tumor-free groups). Expression was determined by qRT-PCR and presented as described in the Materials and Methods (Geometric Mean ± Geometric STD). B) Androgen receptor (AR) mRNA expression in gastrocnemius muscles from Study 1. Statistics: p values provided in Appendix Table S5A-B. Dunnett's multiple comparison test. CEBPδ, n=4, insufficient sample to analyze all tumor-bearing GTx-024-treated animals.


Figure 6A ) ELISA analysis of serum IL - 6 levels in terminal samples from Study 2 . Tumor - free groups ( n = 6 ) , tumor - bearing receiving Vehicle ( n = 7 ) , GTx - 024 ( n = 10 ) , AR - 42 ( n = 8 ) or Combination ( n = 9 ) . ND , not detected . B and C ) Phospho ( p ) STAT3 ( Y705 ) / total ( t ) STAT3 western blot analysis of gastrocnemius tissues from individual animals treated in Study 1 ( B ) and Study 3 ( C ) . Black circle - tumor - free , blue square - tumor - free / GTx - 024 , black triangle - tumor - bearing , blue diamond - tumor / GTx - 024 , red triangle - tumor / AR - 42 , green circle - tumor / combo . D ) Spleen weights normalized to tumor - corrected terminal body weights of mice from Study 2 . Tumor - free groups ( n = 6 ) , tumor - bearing Vehicle ( n = 7 ) , tumor - bearing receiving GTx - 024 ( n = 10 ) , AR - 42 ( n = 9 ) or Combination ( n = 9 ) . E ) ELISA analysis of pSTAT3 ( Y705 ) / tSTAT3 within C - 26 tumors from Study 2 . Tumor - bearing Vehicle - treated ( n = 7 ) , tumor - bearing receiving GTx - 024 , AR - 42 or Combination ( n = 8 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) ELISA analysis of serum IL-6 levels in terminal samples from Study 2. Tumor-free groups (n=6), tumor-bearing receiving Vehicle (n=7), GTx-024 (n=10), AR-42 (n=8) or Combination (n=9). ND, not detected.B and C) Phospho(p)STAT3 (Y705)/total (t)STAT3 western blot analysis of gastrocnemius tissues from individual animals treated in Study 1 (B) and Study 3 (C). Black circle - tumor-free, blue square - tumor-free/GTx-024, black triangle - tumor-bearing, blue diamond - tumor/GTx-024, red triangle - tumor/AR-42, green circle - tumor/combo.D) Spleen weights normalized to tumor-corrected terminal body weights of mice from Study 2. Tumor-free groups (n=6), tumor-bearing Vehicle (n=7), tumor-bearing receiving GTx-024 (n=10), AR-42 (n=9) or Combination (n=9).E) ELISA analysis of pSTAT3(Y705)/tSTAT3 within C-26 tumors from Study 2. Tumor-bearing Vehicle-treated (n=7), tumor-bearing receiving GTx-024, AR-42 or Combination (n=8).


Figure 7A ) Effect of GTx - 024 and AR - 42 monotherapies on cachexia - related differentially regulated genes ( DEGs ) from RNA - seq analyses of Study 1 gastrocnemius muscles . All three panels consist of individual genes plotted with respect to their log2 fold change and - log10 Benjamini - Hochburg adjusted p - values from the comparison of cachexia vs tumor - free controls . Colors of the points reflect the DEG status of each gene for the given comparison . C ) Significance values from GSEA using combined STAT3 gene sets identified in B . Each treatment group is compared to tumor - bearing control ( cachexia ) transcriptomes . D ) Heat map of DEGs within the combined STAT3 gene sets representing mean z scores calculated from normalized RNAseq count data . Tumor - free control ( black checkered ) , GTx - 024 - treated tumor - free ( blue checkered ) , tumor - bearing control ( black ) , GTx - 024 - treated tumor - bearing ( blue ) , AR - 42 - treated tumor - bearing ( red ) and Combination - treated tumor - bearing ( green ) . E ) Results of GSEA using combined ATF - 1 gene sets identified in B across treatment groups versus tumor - bearing control ( cachexia ) transcriptomes . Tumor - free control ( black checkered ) , GTx - 024 - treated tumor - free ( blue checkered ) , tumor - bearing control ( black ) , GTx - 024 - treated tumor - bearing ( blue ) , AR - 42 - treated tumor - bearing ( red ) and Combination - treated tumor - bearing ( green ) . F ) Heat map of DEGs within the combined ATF - 1 gene sets ( mean z score ) . Treatment groups are as in D . G ) mRNA expression of mediators of IL - 6 signaling upstream of STAT3 . Data presented as mean ± SD of per animal log - transformed fold change ( log2FC ) values versus tumor - free controls . Groups : Tumor - free control ( n = 6 ) , tumor - free GTx - 024 treated ( n = 5 ) , tumor - bearing receiving vehicle ( n = 4 ) , GTx - 024 ( n = 5 ) , AR - 42 ( n = 5 ) or Combination ( n = 6 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) Effect of GTx-024 and AR-42 monotherapies on cachexia-related differentially regulated genes (DEGs) from RNA-seq analyses of Study 1 gastrocnemius muscles. All three panels consist of individual genes plotted with respect to their log2 fold change and -log10 Benjamini-Hochburg adjusted p-values from the comparison of cachexia vs tumor-free controls. Colors of the points reflect the DEG status of each gene for the given comparison.C) Significance values from GSEA using combined STAT3 gene sets identified in B. Each treatment group is compared to tumor-bearing control (cachexia) transcriptomes.D) Heat map of DEGs within the combined STAT3 gene sets representing mean z scores calculated from normalized RNAseq count data. Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green).E) Results of GSEA using combined ATF-1 gene sets identified in B across treatment groups versus tumor-bearing control (cachexia) transcriptomes.Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green). F) Heat map of DEGs within the combined ATF-1 gene sets (mean z score). Treatment groups are as in D.G) mRNA expression of mediators of IL-6 signaling upstream of STAT3. Data presented as mean±SD of per animal log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).


Figure 8A ) Volcano plot from RNA - seq analyses of Study 1 gastrocnemius muscles for tumor - bearing combination - treated mice versus tumor - bearing controls . Genes not differentially expressed in this comparison are indicated with blue . The remaining genes ( red and green ) are DEGs in the combination - treated versus tumor - bearing control comparison . The green coloring indicates the subset of these DEGs that are not also differentially expressed in the comparison of AR - 42 - treated tumor - bearing mice versus tumor - bearing controls , suggesting these genes are responsive to only the combination therapy . Log2 - transformed fold change ( FC ) in expression is plotted on the x - axis and - log10 transformed Benjamini - Hochburg adjusted p - values are plotted on the y - axis . C ) Significance values from GSEA using combined CTNNB1 gene sets . Each treatment group was compared to tumor - free control transcriptomes . * FDR < 1e - 5 determined , set to 1e - 5 for plot . D ) Enrichment plots from GSEA of the CTNNB1 gene set for each treatment group versus tumor - free control comparisons . GTx - 024 - treated tumor - free ( blue checkered ) , tumor - bearing control ( black ) , GTx - 024 - treated tumor - bearing ( blue ) , AR - 42 - treated tumor - bearing ( red ) and Combination - treated tumor - bearing ( green ) . E ) mRNA expression of WNT effectors upstream of β - catenin . Data are presented as mean ± SD of log - transformed fold change ( log2FC ) values versus tumor - free controls . Groups : Tumor - free control ( n = 6 ) , tumor - free GTx - 024 treated ( n = 5 ) , tumor - bearing receiving vehicle ( n = 4 ) , GTx - 024 ( n = 5 ) , AR - 42 ( n = 5 ) or Combination ( n = 6 ) .

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Figure 8A) Volcano plot from RNA-seq analyses of Study 1 gastrocnemius muscles for tumor-bearing combination-treated mice versus tumor-bearing controls. Genes not differentially expressed in this comparison are indicated with blue. The remaining genes (red and green) are DEGs in the combination-treated versus tumor-bearing control comparison. The green coloring indicates the subset of these DEGs that are not also differentially expressed in the comparison of AR-42-treated tumor-bearing mice versus tumor-bearing controls, suggesting these genes are responsive to only the combination therapy. Log2-transformed fold change (FC) in expression is plotted on the x-axis and -log10 transformed Benjamini-Hochburg adjusted p-values are plotted on the y-axis.C) Significance values from GSEA using combined CTNNB1 gene sets. Each treatment group was compared to tumor-free control transcriptomes. *FDR < 1e-5 determined, set to 1e-5 for plot.D) Enrichment plots from GSEA of the CTNNB1 gene set for each treatment group versus tumor-free control comparisons. GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green).E) mRNA expression of WNT effectors upstream of β-catenin. Data are presented as mean±SD of log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).


Figure 1A . Immunohistochemical analysis of SHARPIN expression in the human mammary gland . Cross - section of a mammary duct ( upper panel ) and magnification of the marked area ( lower panel ) . SHARPIN - positive luminal ( grey arrow ) and stromal cells ( red arrow ) , and the approximate position of the basal lamina ( dashed red line ) are indicated . Scale bar represents 50 μm . B . Western blot analysis of SHARPIN protein expression in isolated primary mammary epithelial cells ( MECs ) and mammary stromal fibroblasts ( MSFs ) . CDH1 and vimentin were used as markers of epithelial and stromal cell lineages , respectively . GAPDH served as a control for protein loading . C . FACS - based isolation of mouse mammary gland basal epithelial cells ( LinnegCD24intICAM - 1hi ) , mature luminal epithelial cells ( LinnegCD24hiICAM - 1neg ) , luminal progenitor cells ( LinnegCD24hiICAM - 1int ) , and stromal cells ( LinnegCD24neg ) . D . Quantitative PCR analysis of SHARPIN mRNA expression in cell populations isolated in ( C ) ( mean ± SEM , n = 3 - 5 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Immunohistochemical analysis of SHARPIN expression in the human mammary gland. Cross-section of a mammary duct (upper panel) and magnification of the marked area (lower panel). SHARPIN-positive luminal (grey arrow) and stromal cells (red arrow), and the approximate position of the basal lamina (dashed red line) are indicated. Scale bar represents 50 μm.B. Western blot analysis of SHARPIN protein expression in isolated primary mammary epithelial cells (MECs) and mammary stromal fibroblasts (MSFs). CDH1 and vimentin were used as markers of epithelial and stromal cell lineages, respectively. GAPDH served as a control for protein loading.C. FACS-based isolation of mouse mammary gland basal epithelial cells (LinnegCD24intICAM-1hi), mature luminal epithelial cells (LinnegCD24hiICAM-1neg), luminal progenitor cells (LinnegCD24hiICAM-1int), and stromal cells (LinnegCD24neg).D. Quantitative PCR analysis of SHARPIN mRNA expression in cell populations isolated in (C) (mean ± SEM, n = 3 - 5).