Distribution of rotation frequencies in trapped sperm ( n = 32 ; mean frequency ± SD 6 . 0 ± 2 . 1 Hz ) .

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Distribution of rotation frequencies in trapped sperm (n = 32; mean frequency ± SD 6.0 ± 2.1 Hz).


Image series of a sperm cell trapped parallel to the optical axis ; images were obtained at t = 0 , 95 , 165 , and 205 ms . The red bar indicates the 360 ° rotation of the tip of the head . Scale bar represents 10 µm .

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Image series of a sperm cell trapped parallel to the optical axis; images were obtained at t = 0, 95, 165, and 205 ms. The red bar indicates the 360° rotation of the tip of the head. Scale bar represents 10 µm.


Representative distribution of rotation frequencies of freely swimming CatSper - deficient sperm incubated under non - capacitating ( 0 mM bicarbonate ; black ; n = 73 ) and capacitating conditions ( 25 mM bicarbonate ; red ; n = 272 ) .

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Representative distribution of rotation frequencies of freely swimming CatSper-deficient sperm incubated under non-capacitating (0 mM bicarbonate; black; n = 73) and capacitating conditions (25 mM bicarbonate; red; n = 272).


Rolling frequency ( mean ± SD ) of freely swimming CATSPER2 - / - sperm incubated under non - capacitating ( 0 mM bicarbonate , n = 1009 ) and capacitating ( 25 mM bicarbonate , n = 946 ) conditions .

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Rolling frequency (mean ± SD) of freely swimming CATSPER2-/- sperm incubated under non-capacitating (0 mM bicarbonate, n = 1009) and capacitating (25 mM bicarbonate, n = 946) conditions.


Rolling frequency ( mean ± SD ) of freely swimming CATSPER2 - / - sperm in 0 ( n = 457 ) , 0 . 2 ( n = 389 ) , and 1 % ( w / v ) ( n = 187 ) methyl cellulose .

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Rolling frequency (mean ± SD) of freely swimming CATSPER2-/- sperm in 0 (n = 457), 0.2 (n = 389), and 1 % (w/v) (n = 187) methyl cellulose.


Image series of a CATSPER2 - / - sperm cell optically trapped perpendicular to the optical axis ; images were obtained at t = 0 , 75 , 150 , and 225 ms . Scale bar represents 10 µm . Image series of a CATSPER2 - / - sperm cell optically trapped parallel to the optical axis ; images were obtained at t = 0 , 60 , 195 , and 320 ms . The red bar indicates the 360 ° rotation of the tip of the head . Scale bar represents 10 µm . Representative time courses of the rotation frequencies of optically trapped CATSPER2 - / - sperm ( each sperm is represented in a different color ) . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis .

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Image series of a CATSPER2-/- sperm cell optically trapped perpendicular to the optical axis; images were obtained at t = 0, 75, 150, and 225 ms. Scale bar represents 10 µm. Image series of a CATSPER2-/- sperm cell optically trapped parallel to the optical axis; images were obtained at t = 0, 60, 195, and 320 ms. The red bar indicates the 360° rotation of the tip of the head. Scale bar represents 10 µm. Representative time courses of the rotation frequencies of optically trapped CATSPER2-/- sperm (each sperm is represented in a different color). Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis.


Rotation frequency of a trapped sperm cell before and after dragging across the barrier stream . The green trace indicates the fluorescence of fluorescein included in the barrier stream . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis . Paired plot of rotation frequencies of individual sperm cells before and after dragging across the barrier stream .

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Rotation frequency of a trapped sperm cell before and after dragging across the barrier stream. The green trace indicates the fluorescence of fluorescein included in the barrier stream. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies of individual sperm cells before and after dragging across the barrier stream.


Rotation frequency of a trapped sperm cell before and after dragging from the control stream containing 0 mM bicarbonate into the stimulus stream containing 25 mM bicarbonate . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis . Paired plot of rotation frequencies of individual sperm at 0 and 25 mM bicarbonate .

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Rotation frequency of a trapped sperm cell before and after dragging from the control stream containing 0 mM bicarbonate into the stimulus stream containing 25 mM bicarbonate. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies of individual sperm at 0 and 25 mM bicarbonate.


Rotation frequency of a trapped sperm cell in the presence and absence of extracellular Ca2 + . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis . Paired plot of rotation frequencies in the presence and absence of extracellular Ca2 + .

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Rotation frequency of a trapped sperm cell in the presence and absence of extracellular Ca2+. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies in the presence and absence of extracellular Ca2+.


Fraction of motile sperm ( mean ± SD ) in a sperm population incubated in the presence ( black ) and absence ( at t = 0 ) of extracellular Ca2 + ( red ) ( n ≥ 5 ) .

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Fraction of motile sperm (mean ± SD) in a sperm population incubated in the presence (black) and absence (at t = 0) of extracellular Ca2+ (red) (n ≥ 5).


Distribution of rotation frequencies in populations of freely swimming sperm in the presence ( black , n = 335 ) and absence ( red , n = 224 ) of extracellular Ca2 + ( red ) .

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Distribution of rotation frequencies in populations of freely swimming sperm in the presence (black, n = 335) and absence (red, n = 224) of extracellular Ca2+ (red).


Left : Bright - field image series of a freely swimming wild - type sperm cell at t = 0 , 151 , and 303 ms . Scale bar represents 10 µm . Right : Rotation frequency ( mean ± SD ) of 24 freely swimming wild - type sperm . Left : Bright - field image series of a freely swimming Catsper1 - / - sperm cell at t = 0 , 151 , and 294 ms . Scale bar represents 10 µm . Right : Rotation frequency ( mean ± SD ) of 24 freely swimming Catsper1 - / - sperm .

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Left: Bright-field image series of a freely swimming wild-type sperm cell at t = 0, 151, and 303 ms. Scale bar represents 10 µm. Right: Rotation frequency (mean ± SD) of 24 freely swimming wild-type sperm. Left: Bright-field image series of a freely swimming Catsper1-/- sperm cell at t = 0, 151, and 294 ms. Scale bar represents 10 µm. Right: Rotation frequency (mean ± SD) of 24 freely swimming Catsper1-/- sperm.


B - H Live confocal images of ddaC neurons expressing mCD8 - GFP by ppk - Gal4 at WP stage or 16 h APF . Neurons expressing efa6 RNAi # 1 - # 3 ( C - E ) , efa612 mutant MARCM clones ( F ) , and efa612 / GX6w - transheterozygous mutant neurons ( G ) showed consistent dendrite pruning defects at 16 h APF , as compared to the wild - type neurons ( B ) . The dendrite pruning defects were fully rescued when overexpressing full - length Efa6 in the efa612 mutant clones ( H ) . Red arrowheads point to the ddaC somas .

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B-H Live confocal images of ddaC neurons expressing mCD8-GFP by ppk-Gal4 at WP stage or 16 h APF. Neurons expressing efa6 RNAi #1-#3 (C-E), efa612 mutant MARCM clones (F), and efa612/GX6w- transheterozygous mutant neurons (G) showed consistent dendrite pruning defects at 16 h APF, as compared to the wild-type neurons (B). The dendrite pruning defects were fully rescued when overexpressing full-length Efa6 in the efa612 mutant clones (H). Red arrowheads point to the ddaC somas.


Live confocal images of efa612 / GX6w - mutant ddaC neurons at WP stage and 16 h APF . Expression of Efa6FL ( C ) rescued the pruning defects in efa612 / GX6w - mutant ddaC neurons ( B ) . Red arrowheads point to the ddaC somas .

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Live confocal images of efa612/GX6w- mutant ddaC neurons at WP stage and 16 h APF. Expression of Efa6FL (C) rescued the pruning defects in efa612/GX6w- mutant ddaC neurons (B). Red arrowheads point to the ddaC somas.


Live confocal images of efa612 / GX6w - mutant ddaC neurons at WP stage and 16 h APF . Expression of Efa6Nterm ( D ) , Efa6ΔPH ( F ) and Efa6Nterm + CAAX ( H ) , but not Efa6Cterm ( E ) or Efa6ΔMTED ( G ) , rescued the pruning defects in efa612 / GX6w - mutant ddaC neurons Red arrowheads point to the ddaC somas . I - J Quantification of dendrite severing defects and unpruned dendrite lengths at 16 h APF .

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Live confocal images of efa612/GX6w- mutant ddaC neurons at WP stage and 16 h APF. Expression of Efa6Nterm (D), Efa6ΔPH (F) and Efa6Nterm+CAAX (H), but not Efa6Cterm (E) or Efa6ΔMTED (G), rescued the pruning defects in efa612/GX6w- mutant ddaC neurons Red arrowheads point to the ddaC somas. I-J Quantification of dendrite severing defects and unpruned dendrite lengths at 16 h APF.


A - D Live confocal images of ddaC neurons at WP stage and 7 h APF . Efa6FL or Efa6Nterm - overexpressing neurons ( B , C ) showed precocious pruning phenotype at 7 h APF , compared to the control and Efa6ΔMTED - overexpressing neurons ( A , D ) in which most dendrites remained attached to the soma at the same time point . Red arrowheads point to the ddaC somas . Dorsal dendrites are marked by red arrows . E Quantification of precocious pruning defects as indicated by the percentage of dorsal dendrites attached to the soma at 7 h APF .

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A-D Live confocal images of ddaC neurons at WP stage and 7 h APF. Efa6FL or Efa6Nterm-overexpressing neurons (B, C) showed precocious pruning phenotype at 7 h APF, compared to the control and Efa6ΔMTED-overexpressing neurons (A, D) in which most dendrites remained attached to the soma at the same time point. Red arrowheads point to the ddaC somas. Dorsal dendrites are marked by red arrows. E Quantification of precocious pruning defects as indicated by the percentage of dorsal dendrites attached to the soma at 7 h APF.


F - I Confocal images of da sensory neurons expressing Efa6 - Ki - GFP ( F ) , or UAS - Efa6 - GFP driven by ppk - Gal4 ( H ) were co - stained with GFP ( Green ) and Nrg ( Magenta ) at wL3 stage . ( G , I ) Confocal images of the epidermal cells expressing Efa6 - Ki - GFP ( G ) or UAS - Efa6 - GFP driven by A58 - Gal4 ( I ) were co - stained with GFP ( Green ) and Dlg ( Magenta ) at wL3 stage . The ddaC soma is marked by dashed lines .

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F-I Confocal images of da sensory neurons expressing Efa6-Ki-GFP (F), or UAS-Efa6-GFP driven by ppk-Gal4 (H) were co-stained with GFP (Green) and Nrg (Magenta) at wL3 stage. (G, I) Confocal images of the epidermal cells expressing Efa6-Ki-GFP (G) or UAS-Efa6-GFP driven by A58-Gal4 (I) were co-stained with GFP (Green) and Dlg (Magenta) at wL3 stage. The ddaC soma is marked by dashed lines.


A - F Representative kymographs of EB1 - GFP comets in the proximal dendrites of ddaC neurons at 96 h AEL . The horizontal arrow indicates the direction toward the soma , and the vertical arrow indicates the time . In contrast to the wild type ( A ) , the number of EB1 - GFP comets was significantly increased in efa612 / GX6w - mutant neurons ( B ) . The comet length and speed were significantly decreased in Efa6FL - and Efa6Nterm - overexpressing neurons , but not in Efa6ΔMTED - overexpression neurons ( C - F ) .

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A-F Representative kymographs of EB1-GFP comets in the proximal dendrites of ddaC neurons at 96 h AEL. The horizontal arrow indicates the direction toward the soma, and the vertical arrow indicates the time. In contrast to the wild type (A), the number of EB1-GFP comets was significantly increased in efa612/GX6w- mutant neurons (B). The comet length and speed were significantly decreased in Efa6FL- and Efa6Nterm-overexpressing neurons, but not in Efa6ΔMTED-overexpression neurons (C-F).


A - C Live confocal images of ddaC dendrites expressing tdEOS : : α - tubulin driven by ppk - Gal4 . The microtubule turnover rate was significantly reduced in efa6 RNAi neurons at wL3 ( A , right panels ) and WP stages ( B , right panels ) , and increased in Efa6 - overexpressing neurons at wL3 stage ( C , right panels ) , compared to the control neurons ( A - C , left panels ) .

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A-C Live confocal images of ddaC dendrites expressing tdEOS::α-tubulin driven by ppk-Gal4. The microtubule turnover rate was significantly reduced in efa6 RNAi neurons at wL3 (A, right panels) and WP stages (B, right panels), and increased in Efa6-overexpressing neurons at wL3 stage (C, right panels), compared to the control neurons (A-C, left panels).


A - H Live confocal images of ddaC neurons at WP stage and 16 h APF . Treatment with colchicine significantly suppressed the pruning defects of efa6 RNAi neurons ( A , B ) , while taxol treatment enhanced the pruning defects ( C , D ) . Removal of one copy of γTub23C ( F ) or expression of γTub23C RNAi ( H ) almost fully rescued the dendrite pruning defects in efa6 mutant ( E ) or RNAi neurons ( G ) , respectively . Red arrowheads point to the ddaC somas .

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A-H Live confocal images of ddaC neurons at WP stage and 16 h APF. Treatment with colchicine significantly suppressed the pruning defects of efa6 RNAi neurons (A, B), while taxol treatment enhanced the pruning defects (C, D). Removal of one copy of γTub23C (F) or expression of γTub23C RNAi (H) almost fully rescued the dendrite pruning defects in efa6 mutant (E) or RNAi neurons (G), respectively. Red arrowheads point to the ddaC somas.


A - F Live confocal images of ddaC neurons at WP stage and 16 h APF . stai RNAi neurons ( B ) , staiSK7 ( C ) , staiKO ( D ) mutant MARCM clones showed similar dendrite pruning defects , compared to the wild - type ddaC neurons ( A ) . Double RNAi of stai and efa6 significantly enhanced the dendrite pruning defects ( E ) , compared to their individual RNAi knockdown . Expression of stai RNAi in efa612 / GX6w - mutant background also significantly enhanced the dendrite pruning defects ( F ) , compared to the RNAi or mutant alone . Red arrowheads point to the ddaC somas .

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A-F Live confocal images of ddaC neurons at WP stage and 16 h APF. stai RNAi neurons (B), staiSK7 (C), staiKO (D) mutant MARCM clones showed similar dendrite pruning defects, compared to the wild-type ddaC neurons (A). Double RNAi of stai and efa6 significantly enhanced the dendrite pruning defects (E), compared to their individual RNAi knockdown. Expression of stai RNAi in efa612/GX6w- mutant background also significantly enhanced the dendrite pruning defects (F), compared to the RNAi or mutant alone. Red arrowheads point to the ddaC somas.


I - L Representative kymographs of EB1 - GFP comets driven by Gal44 - 77 in the proximal dendrites of ddaC neurons at 96 h AEL .

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I-L Representative kymographs of EB1-GFP comets driven by Gal44-77 in the proximal dendrites of ddaC neurons at 96 h AEL.


D , Western blot showing AGO1x expression in three cell lines . For comparison , a parallel blot was probed with an antibody directed against canonical AGO1 . Tubulin served as loading control .

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D, Western blot showing AGO1x expression in three cell lines. For comparison, a parallel blot was probed with an antibody directed against canonical AGO1. Tubulin served as loading control.


E , F , Annotated MS / MS spectrum of peptides specific for the endogenous AGO1x , " QNAVTSLDR " , depicted in green ( E ) and " LSKPQELCHPNPEEAR " , depicted in blue ( F ) . The Mascot Ion Score ( text color corresponds to peptides marked in Fig 1C for reference ) as well as the annotated fragments ( blue = y - ions ; red = b - ions ) together with the corresponding amino acids are indicated .

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E,F, Annotated MS/MS spectrum of peptides specific for the endogenous AGO1x, "QNAVTSLDR", depicted in green (E) and "LSKPQELCHPNPEEAR", depicted in blue (F). The Mascot Ion Score (text color corresponds to peptides marked in Fig 1C for reference) as well as the annotated fragments (blue = y-ions; red = b-ions) together with the corresponding amino acids are indicated.


A , Representative immunofluorescence images showing the subcellular distribution of AGO1x ( red ) relative to nuclear and cytoplasmic markers . DAPI was used to mark the nucleus ( blue ) . The co - stained subcellular marker is indicated in each panel in green . SC 35 , Lsm4 , α - Tubulin , ERP72 , p54 ( NRB ) , and Nucleolin serve as markers for nuclear speckles , cytosol and nucleus , cytosol , endoplasmic reticulum , paraspeckle and nucleolus , respectively .

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A, Representative immunofluorescence images showing the subcellular distribution of AGO1x (red) relative to nuclear and cytoplasmic markers. DAPI was used to mark the nucleus (blue). The co-stained subcellular marker is indicated in each panel in green. SC 35, Lsm4, α-Tubulin, ERP72, p54 (NRB), and Nucleolin serve as markers for nuclear speckles, cytosol and nucleus, cytosol, endoplasmic reticulum, paraspeckle and nucleolus, respectively.


B , Mean ( + / - s . d . ) pixel intensities of AGO1x staining in nucleolus and nucleoplasm , computed from z - stack images of MDA - MB - 231 cells ( n = 20 ) . The P - value was determined using a paired two - tailed t - test .

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B, Mean (+/- s.d.) pixel intensities of AGO1x staining in nucleolus and nucleoplasm, computed from z-stack images of MDA-MB-231 cells (n=20). The P-value was determined using a paired two-tailed t-test.


C , Representative AGO1x and AGO1 blot from MDA - MB - 231 cell fractions . GAPDH and hnRNP C1 / C2 served as markers of purity of the individual fractions .

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C, Representative AGO1x and AGO1 blot from MDA-MB-231 cell fractions. GAPDH and hnRNP C1/C2 served as markers of purity of the individual fractions.


A , Top : Scheme of the sites targeted by sgRNA in the generation of AGO1x mutant cell lines . Bottom : Western analysis of lysates from mutant MDA - MB - 231 cells showing the depletion of AGO1x and the comparable total AGO1 levels across the cell lines . Three independently collected sets of cells were lysed and analysed in parallel . After calculating the levels of AGO1x and AGO1 relative to GAPDH , values corresponding to individual lanes were normalized to the average over the replicates of the control line . These numbers are indicated below the blots . The P - values of the t - tests comparing the expression AGO1x in mutant ( W1A and W6A ) and control ( CTRL ) lines were W1A - CTRL : 0 . 0008 , W6A - CTRL : 0 . 0002 , and for AGO1 W1A - CTRL : 0 . 1227 , W6A - CTRL : 0 . 1173 .

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A, Top: Scheme of the sites targeted by sgRNA in the generation of AGO1x mutant cell lines. Bottom: Western analysis of lysates from mutant MDA-MB-231 cells showing the depletion of AGO1x and the comparable total AGO1 levels across the cell lines. Three independently collected sets of cells were lysed and analysed in parallel. After calculating the levels of AGO1x and AGO1 relative to GAPDH, values corresponding to individual lanes were normalized to the average over the replicates of the control line. These numbers are indicated below the blots. The P-values of the t-tests comparing the expression AGO1x in mutant (W1A and W6A) and control (CTRL) lines were W1A-CTRL: 0.0008, W6A-CTRL: 0.0002, and for AGO1 W1A-CTRL: 0.1227, W6A-CTRL: 0.1173.


B , Phase contrast images of control and mutant cell lines at 24 and 48 hr after plating equal numbers ( 10 ' 000 ) of cells in individual wells of a six well plate .

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B, Phase contrast images of control and mutant cell lines at 24 and 48 hr after plating equal numbers (10'000) of cells in individual wells of a six well plate.


C , Impedance - based mean ( + / - s . d . ) cell indices at the indicated time points after seeding equal numbers of CTRL ( n = 6 ) , W1A ( n = 5 ) and W6A ( n = 5 ) cells . From 6 hours on , there is a statistically significant difference between control and the two mutant cell lines ( P < 0 . 001 , two tailed t - test ) . D , Impedance - based mean ( + / - s . d . ) cell indices as a function of time for control ( n = 3 ) , W1A ( n = 4 ) and W6A ( n = 3 ) cells grown in electronically monitored Boyden chambers . From 12 hours on , there is a statistical significant difference between control and the two mutants ( P < 0 . 005 , two tailed t - test ) .

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C, Impedance-based mean (+/- s.d.) cell indices at the indicated time points after seeding equal numbers of CTRL (n=6), W1A (n=5) and W6A (n=5) cells. From 6 hours on, there is a statistically significant difference between control and the two mutant cell lines (P < 0.001, two tailed t-test). D, Impedance-based mean (+/- s.d.) cell indices as a function of time for control (n=3), W1A (n=4) and W6A (n=3) cells grown in electronically monitored Boyden chambers. From 12 hours on, there is a statistical significant difference between control and the two mutants (P < 0.005, two tailed t-test).


E , Mean ( + / - s . d . ) number of colonies obtained in a soft agar colony formation assay of control , W1A and W6A mutant cell lines ( all n = 4 , P - value determined using an unpaired two - tailed t - test ) . F , Mean ( + / - s . d . ) number of spheres obtained from a sphere formation assay of control , W1A and W6A mutant cell lines ( all n = 8 , P - values computed as in E ) .

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E, Mean (+/- s.d.) number of colonies obtained in a soft agar colony formation assay of control, W1A and W6A mutant cell lines (all n=4, P-value determined using an unpaired two-tailed t-test). F, Mean (+/- s.d.) number of spheres obtained from a sphere formation assay of control, W1A and W6A mutant cell lines (all n=8, P-values computed as in E).


G , AGO1x staining of tissue sections from breast cancers , classified as low - proliferating or high - proliferating based on Ki - 67 staining . The upper and lower panel represents two different tissue sections . H , Mean ( + / - s . d . ) percentage of AGO1x positive cells in breast tumors with low ( n = 7 ) and high ( n = 9 ) proliferation index , assessed by the expression level of Ki - 67 . P - value was determined using an unpaired two - tailed t - test .

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

G, AGO1x staining of tissue sections from breast cancers, classified as low-proliferating or high-proliferating based on Ki-67 staining. The upper and lower panel represents two different tissue sections. H, Mean (+/- s.d.) percentage of AGO1x positive cells in breast tumors with low (n=7) and high (n=9) proliferation index, assessed by the expression level of Ki-67. P-value was determined using an unpaired two-tailed t-test.


A , Scatter plot of mean log2 mRNA fold - changes in the mutant cell lines versus control ( n = 3 ) . Shown are also the Pearson correlation coefficient and respective P - value . The dashed line indicates equal change in the two mutant lines .

O O O O O O O O O O O O O O O O O O O O O O O O O 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, Scatter plot of mean log2 mRNA fold-changes in the mutant cell lines versus control (n=3). Shown are also the Pearson correlation coefficient and respective P-value. The dashed line indicates equal change in the two mutant lines.


C , Mean ( + / - s . d . ) activity of STAT1 / IRF1 / IRF2 / IRF3 / IRF9 transcription factor motifs estimated by ISMARA ( Balwierz et al , 2014 ) ( n = 3 ) . Shown are the P - values ( * * * P < 0 . 001 ) determined by Dunn ' s multiple comparisons test post hoc and the non - parametric Kruskal - Wallis test ( P < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Mean (+/- s.d.) activity of STAT1/IRF1/IRF2/IRF3/IRF9 transcription factor motifs estimated by ISMARA (Balwierz et al, 2014) (n=3). Shown are the P-values (*** P < 0.001) determined by Dunn's multiple comparisons test post hoc and the non-parametric Kruskal-Wallis test (P < 0.0001).


D , Mean ( + / - s . e . m . ) log2 mRNA expression fold - changes ( inferred from RNA - Seq ) of genes involved in the interferon alpha response and apoptosis in the two mutant cell lines relative to control ( n = 3 ) . Multiple - testing - corrected P - values for Wald tests comparing fold - changes with respect to control are depicted above each bar ( * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Mean (+/- s.e.m.) log2 mRNA expression fold-changes (inferred from RNA-Seq) of genes involved in the interferon alpha response and apoptosis in the two mutant cell lines relative to control (n=3). Multiple-testing-corrected P-values for Wald tests comparing fold-changes with respect to control are depicted above each bar (** P < 0.01, *** P < 0.001).


E , Mean ( + / - s . d . ) expression fold - changes of the corresponding proteins ( if detected in the proteomics data ) in the two mutant cell lines ( W1A , n = 2 ; W6A , n = 1 ) compared to Control ( n = 2 ) . Multiple - testing - corrected P - values for unpaired two - tailed t - tests ( proteomics ) comparing fold - changes with respect to control are depicted above each bar ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Mean (+/- s.d.) expression fold-changes of the corresponding proteins (if detected in the proteomics data) in the two mutant cell lines (W1A, n=2; W6A, n=1) compared to Control (n=2). Multiple-testing-corrected P-values for unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (* P < 0.05, ** P < 0.01, *** P < 0.001).


F , Representative result of the apoptosis assay using AnnexinV and propidium iodide ( PI ) staining in the control and the two mutant cell lines ( left ) . The percentage of cells in each quadrant is depicted for each cell line . Quantification of the mean ( + / - s . d . ) percentage of apoptotic cells ( Q1 + Q2 , AnnexinV + ) across the different cell lines ( n = 4 ) ( right ) . Shown is the P - value determined by unpaired two - tailed t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative result of the apoptosis assay using AnnexinV and propidium iodide (PI) staining in the control and the two mutant cell lines (left). The percentage of cells in each quadrant is depicted for each cell line. Quantification of the mean (+/- s.d.) percentage of apoptotic cells (Q1+Q2, AnnexinV+) across the different cell lines (n=4) (right). Shown is the P-value determined by unpaired two-tailed t-test.


G , Impedance - based mean ( + / - s . d . ) cell index values at the indicated time points of growth after seeding equal numbers of control , W1A and W6A cells , upon treatment with DMSO or Ruxolitinib . For the DMSO treatment : Control ( n = 4 ) , W1A ( n = 3 ) and W6A ( n = 3 ) . For the Ruxolitinib treatment : Control ( n = 6 ) , W1A ( n = 3 ) and W6A ( n = 4 ) . From 6 hours after DMSO treatment , there is a statistically significant difference between control and the two mutants ( P < 0 . 05 , two tailed t - test ) , whereas no statistically significant difference is found after Ruxolitinib treatment .

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

G, Impedance-based mean (+/- s.d.) cell index values at the indicated time points of growth after seeding equal numbers of control, W1A and W6A cells, upon treatment with DMSO or Ruxolitinib. For the DMSO treatment: Control (n=4), W1A (n=3) and W6A (n=3). For the Ruxolitinib treatment: Control (n=6), W1A (n=3) and W6A (n=4). From 6 hours after DMSO treatment, there is a statistically significant difference between control and the two mutants (P < 0.05, two tailed t-test), whereas no statistically significant difference is found after Ruxolitinib treatment.


B , Mean ( + / - s . e . m . ) log2 mRNA fold - changes of dsRNAs sensors ( from panel A ) in the two mutant cell lines compared to control ( n = 3 ) inferred from RNA - Seq . Multiple - testing - corrected P - values for Wald tests comparing fold - changes with respect to control are depicted above each bar ( * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Mean (+/- s.e.m.) log2 mRNA fold-changes of dsRNAs sensors (from panel A) in the two mutant cell lines compared to control (n=3) inferred from RNA-Seq. Multiple-testing-corrected P-values for Wald tests comparing fold-changes with respect to control are depicted above each bar (*** P < 0.001).


C , Mean ( + / - s . d . ) fold - changes of corresponding proteins ( see B ) , if detected in the proteomics data from the two mutant ( W1A , n = 2 ; W6A , n = 1 ) lines relative to control ( n = 2 ) . Multiple - testing - corrected P - values for unpaired two - tailed t - tests ( proteomics ) comparing fold - changes with respect to control are depicted above each bar ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

C, Mean (+/- s.d.) fold-changes of corresponding proteins (see B), if detected in the proteomics data from the two mutant (W1A, n=2; W6A, n=1) lines relative to control (n=2). Multiple-testing-corrected P-values for unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (* P < 0.05, ** P < 0.01, *** P < 0.001).


D , Western blot detection of phosphorylated eIF2α ( Ser51 ) and total eIF2α levels in cell lysates of control and mutant MDA - MB - 231 cells . GAPDH and Tubulin serve as loading control for phosphorylated eIF2α and total eIF2α blots respectively . Numbers indicated below the blots represent the band intensities of target proteins normalized to the loading controls , with the value in the control line taken as baseline of 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Western blot detection of phosphorylated eIF2α (Ser51) and total eIF2α levels in cell lysates of control and mutant MDA-MB-231 cells. GAPDH and Tubulin serve as loading control for phosphorylated eIF2α and total eIF2α blots respectively. Numbers indicated below the blots represent the band intensities of target proteins normalized to the loading controls, with the value in the control line taken as baseline of 1.


E , Mean ( + / - s . d . ) fold - changes in phosphorylated peptide ( phosphorylation sites are indicated in brackets ) levels derived from a proteomic analysis of phosphopeptides in the two mutant lines ( W1A , n = 2 ; W6A , n = 2 ) compared to control ( n = 1 ) . Multiple - testing - corrected P - values for Wald tests ( transcriptomics ) or unpaired two - tailed t - tests ( proteomics ) comparing fold - changes with respect to control are depicted above each bar ( * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Mean (+/- s.d.) fold-changes in phosphorylated peptide (phosphorylation sites are indicated in brackets) levels derived from a proteomic analysis of phosphopeptides in the two mutant lines (W1A, n=2; W6A, n=2) compared to control (n=1). Multiple-testing-corrected P-values for Wald tests (transcriptomics) or unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (** P < 0.01).


F , Quantification of the relative abundance ( mean + / - s . e . m . ) of distinct dsRNA foci in each cell type . 3D images were projected on a single plane , non - overlapping foci were counted from 100 cells of each type , and the numbers in three abundance bins ( < 1 , 1 - 5 , > 5 ) are shown .

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

F, Quantification of the relative abundance (mean+/- s.e.m.) of distinct dsRNA foci in each cell type . 3D images were projected on a single plane, non-overlapping foci were counted from 100 cells of each type, and the numbers in three abundance bins (<1, 1-5, >5) are shown.


G , Representative immunofluorescence image of the control and mutant MDA - MB - 231 cells stained with AGO1x ( green ) and J2 antibody ( red ) and DAPI ( blue ) to mark the nucleus . Loss of AGO1x signal in mutants also confirms the efficacy of CRISPR - induced mutations .

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

G, Representative immunofluorescence image of the control and mutant MDA-MB-231 cells stained with AGO1x (green) and J2 antibody (red) and DAPI (blue) to mark the nucleus. Loss of AGO1x signal in mutants also confirms the efficacy of CRISPR-induced mutations.


Concentration of recovered RNA ( mean + / - s . e . m . ) measured by Nanodrop ™ ( H ) Multiple testing corrected P - values from unpaired two - tailed t - tests are depicted above each comparison ( * P < 0 . 05 , * * * P < 0 . 001 ) .

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

Concentration of recovered RNA (mean +/- s.e.m.) measured by Nanodrop™ (H) Multiple testing corrected P-values from unpaired two-tailed t-tests are depicted above each comparison (* P < 0.05, *** P < 0.001).


relative abundance of various RNA species ( mean + / - s . d . ) in J2 antibody immunoprecipitates from three biological replicates of control and mutant cell lines ( I ) . Multiple testing corrected P - values from unpaired two - tailed t - tests are depicted above each comparison ( * P < 0 . 05 , * * * P < 0 . 001 ) .

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

relative abundance of various RNA species (mean +/- s.d.) in J2 antibody immunoprecipitates from three biological replicates of control and mutant cell lines (I). Multiple testing corrected P-values from unpaired two-tailed t-tests are depicted above each comparison (* P < 0.05, *** P < 0.001).


K , Boxplots showing the proportion of G / C nucleotides in all genes and in genes depleted / enriched in dsRNA - seq compared to total RNA - seq ( n = 3 ) . Shown are the P - values in the non - parametric Mann - Whitney U test ( * * * P < 0 . 001 ) . Boxes extend from the 25th to 75th percentiles ( inter - quartile range ( IQR ) ) , horizontal lines represent the median , whiskers indicate the lowest and highest datum within 1 . 5 * IQR from the lower and upper quartiles , respectively .

K, Boxplots showing the proportion of G/C nucleotides in all genes and in genes depleted/enriched in dsRNA-seq compared to total RNA-seq (n=3). Shown are the P-values in the non-parametric Mann-Whitney U test (*** P < 0.001). Boxes extend from the 25th to 75th percentiles (inter-quartile range (IQR)), horizontal lines represent the median, whiskers indicate the lowest and highest datum within 1.5*IQR from the lower and upper quartiles, respectively.


L , Left : Quantification of transcript abundance ( mean + / - s . e . m . ) by qRT - PCR in AGO1x ( black , n = 6 for putative targets , n = 3 for negative controls ) or IgG ( orange , n = 2 ) IP from control cells . n . d . , not detected . Multiple test corrected P - values for an unpaired two - tailed t - test are depicted above each comparison ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Right : Concentration of RNA ( mean + / - s . d . ) obtained with each type of antibody , with P - value from an unpaired two - tailed Student ' s t - test .

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

L, Left: Quantification of transcript abundance (mean +/- s.e.m.) by qRT-PCR in AGO1x (black, n=6 for putative targets, n=3 for negative controls) or IgG (orange, n=2) IP from control cells. n.d., not detected. Multiple test corrected P-values for an unpaired two-tailed t-test are depicted above each comparison (* P < 0.05, ** P < 0.01, *** P < 0.001). Right: Concentration of RNA (mean +/- s.d.) obtained with each type of antibody, with P-value from an unpaired two-tailed Student's t-test.


B , Western blot analysis to validate the interaction of AGO1x with PNPT1 and DHX9 in control MDA - MB - 231cells . AGO1x pulldowns were compared to IgG pulldowns to verify the relative levels of PNPT1 and DHX9 from four independent biological replicates . The immunoglobulin heavy chain band served as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Western blot analysis to validate the interaction of AGO1x with PNPT1 and DHX9 in control MDA-MB-231cells. AGO1x pulldowns were compared to IgG pulldowns to verify the relative levels of PNPT1 and DHX9 from four independent biological replicates. The immunoglobulin heavy chain band served as a loading control.


Immunoprecipitation of PNPT1 from control and mutant MDA - MB - 231 cells and western blotting to validate the specificity of PNPT1 antibody in comparison to IgG control ( C ) .

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

Immunoprecipitation of PNPT1 from control and mutant MDA-MB-231 cells and western blotting to validate the specificity of PNPT1 antibody in comparison to IgG control (C).


Validation of AGO1x - PNPT1 interaction by reciprocal pulldown . Presence of AGO1x and Fibrillarin in PNPT1 immunoprecipitates . Three independent biological replicates were used , and the eluted proteins were analyzed by western blotting ( D ) .

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

Validation of AGO1x-PNPT1 interaction by reciprocal pulldown. Presence of AGO1x and Fibrillarin in PNPT1 immunoprecipitates. Three independent biological replicates were used, and the eluted proteins were analyzed by western blotting (D).


Western blot analysis to verify the depletion of PNPT1 upon siRNA treatment ( F )

Western blot analysis to verify the depletion of PNPT1 upon siRNA treatment (F)


a representative immunofluorescence image of siPNPT1 / siControl - treated control and W6A mutant MDA - MB - 231 cells stained with J2 antibody ( red ) . DAPI was used to mark the nucleus ( blue ) ( G ) . The right - most panels show a magnification of the cells enclosed by the white boxes in the middle panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 representative immunofluorescence image of siPNPT1/siControl-treated control and W6A mutant MDA-MB-231 cells stained with J2 antibody (red). DAPI was used to mark the nucleus (blue) (G). The right-most panels show a magnification of the cells enclosed by the white boxes in the middle panels.


Fluorescent and histological analysis of liver morphology in WT and yap - / - mutant larvae on a lf : CFP background at 5 dpf . The blue colour overlay represents the fluorescent liver tissue . Scale bar : 200μm ( bright field , BF ) and 50μm ( histology , H + E ) Quantitative analysis of fluorescent liver area in WT and lf : Yap larvae at 5 dpf . n > 30 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± standard error of the mean ( s . e . m . )

Fluorescent and histological analysis of liver morphology in WT and yap-/- mutant larvae on a lf:CFP background at 5 dpf. The blue colour overlay represents the fluorescent liver tissue. Scale bar: 200μm (bright field, BF) and 50μm (histology, H+E) Quantitative analysis of fluorescent liver area in WT and lf:Yap larvae at 5 dpf. n>30, ****p<0.0001, two-sided Student's t-test; values represent the mean ± standard error of the mean (s.e.m.)


Whole - mount in situ hybridization ( WISH ) analysis of progenitor marker prox1 , hhex , and foxa3 expression in WT and yap - / - mutant embryos at 36 hpf . Arrow and bracket highlight the hepatic bud . Scale bar :

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

Whole-mount in situ hybridization (WISH) analysis of progenitor marker prox1, hhex, and foxa3 expression in WT and yap-/- mutant embryos at 36 hpf. Arrow and bracket highlight the hepatic bud. Scale bar: 200μm


qPCR analysis of hhex and prox1 gene expression in WT and yap - / - mutant embryos at 48 hpf . n > 3 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

qPCR analysis of hhex and prox1 gene expression in WT and yap-/- mutant embryos at 48 hpf. n>3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


WISH analysis of prox1 , hhex and foxa3 expression in WT , hs : dnYap and hs : Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf . Arrow and bracket highlight the hepatic bud . Scale bar :

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

WISH analysis of prox1, hhex and foxa3 expression in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf. Arrow and bracket highlight the hepatic bud. Scale bar: 200μm


Quantitative analysis of hepatic bud area ( prox1 staining ) in WT , hs : dnYap and hs : Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf . n > 10 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

Quantitative analysis of hepatic bud area (prox1 staining) in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf. n>10, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


Fluorescent and histological analysis of liver morphology in dissected WT and yap - / - mutant adults on a lf : CFP background . The blue colour overlay represents the fluorescent liver tissue . Scale bar : 2mm ( brightfield , BF ) and 50μm ( histology , H + E )

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

Fluorescent and histological analysis of liver morphology in dissected WT and yap-/- mutant adults on a lf:CFP background. The blue colour overlay represents the fluorescent liver tissue. Scale bar: 2mm (brightfield, BF) and 50μm (histology, H+E)


Quantitative determination of body mass in WT and yap - / - adults . n = 8 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e . m Quantitative determination of liver size in WT and yap - / - adults as determined by measuring liver mass as a percentage of body mass . n = 12 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Quantitative determination of body mass in WT and yap-/- adults. n=8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m Quantitative determination of liver size in WT and yap-/- adults as determined by measuring liver mass as a percentage of body mass. n=12, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


qPCR analysis of the hhex expression in adult liver tissue isolated from WT and yap - / - fish . n > 3 , * * p < 0 . 01 , two - sided Student ' s t - test ; values represent the mean ± s . e . m qPCR analysis of the prox1 expression in adult liver tissue isolated from WT and yap - / - mutant fish . n > 3 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

qPCR analysis of the hhex expression in adult liver tissue isolated from WT and yap-/- fish. n>3, **p<0.01, two-sided Student's t-test; values represent the mean ± s.e.m qPCR analysis of the prox1 expression in adult liver tissue isolated from WT and yap-/- mutant fish. n>3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


Heatmap of RNAseq analysis illustrating statistically significant differential gene expression between WT and yap - / - mutant larvae at 3 dpf . Blue indicates decreased expression , while red demonstrates increased

Heatmap of RNAseq analysis illustrating statistically significant differential gene expression between WT and yap-/- mutant larvae at 3 dpf. Blue indicates decreased expression, while red demonstrates increased expression


Heatmap analysis of RNAseq data highlights the reduction of glucose transporters ( glut1 and glut2 ) in yap - / - mutant larvae at 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

Heatmap analysis of RNAseq data highlights the reduction of glucose transporters (glut1 and glut2) in yap-/- mutant larvae at 3 dpf


qPCR analysis of glut1 and glut2 expression in WT and yap - / - mutant larvae at 3 dpf . n > 3 , * p < 0 . 05 , * * * p < 0 . 001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

qPCR analysis of glut1 and glut2 expression in WT and yap-/- mutant larvae at 3 dpf. n>3, *p<0.05, ***p<0.001, two-sided Student's t-test; values represent the mean ± s.e.m


qPCR analysis of glut1 and glut2 expression in WT , hs : dnYap and hs : Yap transgenic embryos heat shocked at 24 hpf and isolated at 30 hpf . n > 3 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

qPCR analysis of glut1 and glut2 expression in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and isolated at 30 hpf. n>3, *p<0.05, **p<0.01, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m


qPCR analysis of glut1 and glut2 expression in liver tissue dissected from WT , yap - / - mutant and lf : Yap transgenic adults . n > 3 , * p < 0 . 05 , * * p < 0 . 01 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

qPCR analysis of glut1 and glut2 expression in liver tissue dissected from WT, yap-/- mutant and lf:Yap transgenic adults. n>3, *p<0.05, **p<0.01, two-sided Student's t-test; values represent the mean ± s.e.m


Glucose tolerance test in 3 - month old WT and yap - / - mutant zebrafish . Blood glucose was measured 0 , 30 , 60 , 120 , 180 and 240 min post glucose injection . n > 8 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e . m Glucose tolerance test in 12 - month old WT and yap - / - mutant zebrafish . Blood glucose was measured 0 , 30 , 60 , 120 , 180 and 240 min post glucose injection . n > 8 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Glucose tolerance test in 3-month old WT and yap-/- mutant zebrafish. Blood glucose was measured 0, 30, 60, 120, 180 and 240 min post glucose injection. n>8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m Glucose tolerance test in 12-month old WT and yap-/- mutant zebrafish. Blood glucose was measured 0, 30, 60, 120, 180 and 240 min post glucose injection. n>8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


Clustergram analysis of polar metabolite abundance from WT and yap - / - mutant larvae at 3 dpf as determined by LC - MS / MS via selected reaction monitoring ( SRM ) analysis . n = 4 , p < 0 . 05 , two - sided Student ' s t -

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Clustergram analysis of polar metabolite abundance from WT and yap-/- mutant larvae at 3 dpf as determined by LC-MS/MS via selected reaction monitoring (SRM) analysis. n=4, p<0.05, two-sided Student's t-test


Steady state abundance of glycolytic intermediates in WT and yap - / - mutant larvae as determined by SRM analysis . n = 4 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e . m Steady state abundance of nucleotides in WT and yap - / - mutant larvae as determined by SRM analysis . n = 4 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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Steady state abundance of glycolytic intermediates in WT and yap-/- mutant larvae as determined by SRM analysis. n=4, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m Steady state abundance of nucleotides in WT and yap-/- mutant larvae as determined by SRM analysis. n=4, *p<0.05, ***p<0.001, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m


Relative isotopic enrichment of 13C in glycolytic intermediates following incubation of WT and yap - / - mutant larvae in [ U13C6 ] - glucose as determined by SRM analysis . n = 3 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e . m Relative isotopic enrichment of 13C in nucleotide precursors following incubation of WT and yap - / - larvae in [ U13C6 ] - glucose as determined by SRM analysis . n = 3 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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Relative isotopic enrichment of 13C in glycolytic intermediates following incubation of WT and yap-/- mutant larvae in [U13C6]-glucose as determined by SRM analysis. n=3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m Relative isotopic enrichment of 13C in nucleotide precursors following incubation of WT and yap-/- larvae in [U13C6]-glucose as determined by SRM analysis. n=3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


Effect of nucleoside ( NS ) cocktail treatment from 1 - 3 dpf on the incidence of cardiac edema in WT and yap - / - mutant larvae . Each point represents the percentage of affected embryos in each independent clutch . n > 7 , * * p < 0 . 01 , two - sided Student ' s t - test ; values represent the mean s . e .

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Effect of nucleoside (NS) cocktail treatment from 1-3 dpf on the incidence of cardiac edema in WT and yap-/- mutant larvae. Each point represents the percentage of affected embryos in each independent clutch. n>7, **p<0.01, two-sided Student's t-test; values represent the mean s.e.m


Effect of WZB - 117 ( WZB , 10 μM ) treatment from 3 - 5 dpf in WT and lf : Yap larvae on liver size as determined by fluorescence microscopy at 5 dpf in lf : GFP reporters . Scale bar :

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

Effect of WZB-117 (WZB, 10 μM) treatment from 3-5 dpf in WT and lf:Yap larvae on liver size as determined by fluorescence microscopy at 5 dpf in lf:GFP reporters. Scale bar: 200μm


Quantitative analysis of the effect of WZB treatment from 3 - 5 dpf on liver size as determined by fluorescence microscopy . n > 21 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Quantitative analysis of the effect of WZB treatment from 3-5 dpf on liver size as determined by fluorescence microscopy. n>21, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m


Quantitative analysis of the effect of Trehalose ( 100 μM ) treatment from 3 - 5 dpf on liver size as determined by fluorescence microscopy . n > 70 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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Quantitative analysis of the effect of Trehalose (100 μM) treatment from 3-5 dpf on liver size as determined by fluorescence microscopy. n>70, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m


Dot plots showing the correlation between GLUT1 and YAP1 mRNA expression in the cancer cell line encyclopedia ( CCLE , 967 lines ) . Pearson coefficient is 0 . 46 Dot plots showing the correlation between GLUT1 and AMOTL2 mRNA expression in the cancer cell line encyclopedia ( CCLE , 967 lines ) . Pearson coefficient is 0 .

Dot plots showing the correlation between GLUT1 and YAP1 mRNA expression in the cancer cell line encyclopedia (CCLE, 967 lines). Pearson coefficient is 0.46 Dot plots showing the correlation between GLUT1 and AMOTL2 mRNA expression in the cancer cell line encyclopedia (CCLE, 967 lines). Pearson coefficient is 0.45


ChIPseq tracks at the GLUT1 locus highlighting YAP and TEAD enrichment overlaying H3K27Ac marks at an intragenic enhancer element in different contexts ( LATS2 mutant MSTO211H cancer cells and HA - YAP5SA overexpressing HuCCT1 liver cancer cells )

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ChIPseq tracks at the GLUT1 locus highlighting YAP and TEAD enrichment overlaying H3K27Ac marks at an intragenic enhancer element in different contexts (LATS2 mutant MSTO211H cancer cells and HA-YAP5SA overexpressing HuCCT1 liver cancer cells)


Gross morphology of the liver from WT and TetONYap transgenic mice , treated with doxycycline for 10 days . Scale bar : 1

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Gross morphology of the liver from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Scale bar: 1 cm


Immunohistochemical analysis of GLUT1 expression in liver tissue from WT and TetONYap transgenic mice , treated with doxycycline for 10 days . Scale bar :

Immunohistochemical analysis of GLUT1 expression in liver tissue from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Scale bar: 50μm


FDG PET imaging of coronal views from WT and TetONYap transgenic mice , treated with doxycycline for 10 days . Arrow is pointing to the

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FDG PET imaging of coronal views from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Arrow is pointing to the liver


Scintillation analysis of FDG uptake ( fraction of injected dose per gram of liver tissue , % ID / g ) in dissected liver tissue from WT and TetON Yap transgenic mice , treated with doxycycline for 10 days . n = 4 , * * * p < 0 . 001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Scintillation analysis of FDG uptake (fraction of injected dose per gram of liver tissue, %ID/g) in dissected liver tissue from WT and TetON Yap transgenic mice, treated with doxycycline for 10 days. n=4, ***p<0.001, two-sided Student's t-test; values represent the mean ± s.e.m


D : Analytical ultracentrifugation absorbance data analysis of N - NTD . The clear peak at ~ 15 . 4 kDa molecular weight ( MW ) corresponds to the N - NTD monomer . E : Analytical ultracentrifugation absorbance data analysis of N - CTD . The clear peak at ~ 28 . 7 kDa MW corresponds to the N - CTD dimer .

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

D: Analytical ultracentrifugation absorbance data analysis of N-NTD. The clear peak at ~15.4 kDa molecular weight (MW) corresponds to the N-NTD monomer. E: Analytical ultracentrifugation absorbance data analysis of N-CTD. The clear peak at ~28.7 kDa MW corresponds to the N-CTD dimer.


( A ) Immunofluorescence microscopy for POMC ( green ) and Cre ( red ; n = 4 ) , ( B ) POMC ( green ) and Atg7 ( red ; n = 4 ) and ( C ) POMC ( green ) and p62 ( red ; n = 6 ) in MBH sections from Con and Atg7F / F ‐ POMC ‐ Cre ( KO )

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(A) Immunofluorescence microscopy for POMC (green) and Cre (red; n=4), (B) POMC (green) and Atg7 (red; n=4) and (C) POMC (green) and p62 (red; n=6) in MBH sections from Con and Atg7F/F‐POMC‐Cre (KO) mice


( D ) TUNEL ‐ positivity ( green ; n = 4 ) and ( E ) number of POMC ‐ positive neurons ( red ) in MBH sections from Con and KO mice ( n = 6 ) . Values are mean ± s . e . m . P values are as compared with diet ‐ and age ‐ matched controls . * P0 . 05 , * * * P0 . 001 . Nuclei are blue ( DAPI ) . Scale inset : 10 μm . White arrows indicate Atg7 , Cre , p62 or POMC signal . Yellow arrows indicate merged signal . Con , control ; DAPI , 4 ′ ‐ 6 ‐ diamidino ‐ 2 ‐ phenylindole ; KO , knockout ; MBH , mediobasal hypothalamic ; NS , not statistically significant ; POMC , proopiomelanocortin ; TUNEL , TdT ‐ mediated dUTP nick end labelling .

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(D) TUNEL‐positivity (green; n=4) and (E) number of POMC‐positive neurons (red) in MBH sections from Con and KO mice (n=6). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, ***P0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. White arrows indicate Atg7, Cre, p62 or POMC signal. Yellow arrows indicate merged signal. Con, control; DAPI, 4′‐6‐diamidino‐2‐phenylindole; KO, knockout; MBH, mediobasal hypothalamic; NS, not statistically significant; POMC, proopiomelanocortin; TUNEL, TdT‐mediated dUTP nick end labelling.


( A ) Body wt of 4 ‐ mo ‐ and 12 ‐ mo ‐ old Con and KO mice on RD ( n = 9 - 13 ) or HFD ( n = 5 - 7 ) . ( B ) Total body fat and ( C ) lean mass of 4 ‐ mo ‐ old Con and KO mice on RD ( n = 7 ) and 12 ‐ mo ‐ old Con and KO mice on HFD ( n = 6 - 8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Body wt of 4‐mo‐ and 12‐mo‐old Con and KO mice on RD (n=9-13) or HFD (n=5-7). (B) Total body fat and (C) lean mass of 4‐mo‐old Con and KO mice on RD (n=7) and 12‐mo‐old Con and KO mice on HFD (n=6-8).


( D ) eWAT wt , ( E ) fat pads and ( F ) liver wt of 4 ‐ mo ‐ old Con and KO mice on RD ( n = 6 - 8 ) .

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(D) eWAT wt, (E) fat pads and (F) liver wt of 4‐mo‐old Con and KO mice on RD (n=6-8).


( G ) Immunoblots for POMC and ACTH , and ( H ) NPY in MBH from RD ‐ fed Con and KO mice ( n = 3 - 4 ) .

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(G) Immunoblots for POMC and ACTH, and (H) NPY in MBH from RD‐fed Con and KO mice (n=3-4).


( I ) Immunofluorescence microscopy for POMC ( green ) and α ‐ MSH ( red ) , and ( J ) α ‐ MSH ( green ) in MBH from Con and KO mice on RD ( n = 5 ) . Values are mean ± s . e . m . P values are as compared with diet ‐ and age ‐ matched controls . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . Nuclei are blue ( DAPI ) . Scale inset : 10 μm . Yellow arrows indicate merged signal . ACTH , adrenocorticotrophic hormone ; Con , control ; DAPI , 4 ′ ‐ 6 ‐ diamidino ‐ 2 ‐ phenylindole ; eWAT , epididymal fat ; HDF , high ‐ fat diet ; KO , knockout ; MBH , mediobasal hypothalamic ; mo , month ; MSH , melanocyte ‐ stimulating hormone ; NPY , neuropeptide Y ; POMC , proopiomelanocortin ; RD , regular chow ; wt , weight .

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

(I) Immunofluorescence microscopy for POMC (green) and α‐MSH (red), and (J) α‐MSH (green) in MBH from Con and KO mice on RD (n=5). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. Yellow arrows indicate merged signal. ACTH, adrenocorticotrophic hormone; Con, control; DAPI, 4′‐6‐diamidino‐2‐phenylindole; eWAT, epididymal fat; HDF, high‐fat diet; KO, knockout; MBH, mediobasal hypothalamic; mo, month; MSH, melanocyte‐stimulating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin; RD, regular chow; wt, weight.


( A ) Percent loss of body fat and ( B ) lean mass in 24 ‐ h ‐ fasted Con and KO mice on RD or HFD ( n = 6 - 8 ) .

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(A) Percent loss of body fat and (B) lean mass in 24‐h‐fasted Con and KO mice on RD or HFD (n=6-8).


( C ) Histology for eWAT from fed or 6 ‐ h ‐ fasted Con and KO mice on RD ( n = 3 - 4 ) .

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(C) Histology for eWAT from fed or 6‐h‐fasted Con and KO mice on RD (n=3-4).


( D ) Serum fatty acids and ( E ) glycerol from fed or 6 ‐ h ‐ fasted Con and KO mice on RD ( n = 7 - 9 ) .

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(D) Serum fatty acids and (E) glycerol from fed or 6‐h‐fasted Con and KO mice on RD (n=7-9).


( F ) Serum fatty acids and ( G ) glycerol after intraperitoneal isoproterenol ( Iso ) in 6 ‐ h ‐ fasted Con and KO mice on RD ( n = 5 - 6 ) .

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(F) Serum fatty acids and (G) glycerol after intraperitoneal isoproterenol (Iso) in 6‐h‐fasted Con and KO mice on RD (n=5-6).


( H ) Immunoblot for oxidized proteins in eWAT from RD ‐ fed Con and KO mice ( n = 3 ) . ( I ) FACS analysis for M1 and M2 macrophage proportions in adipose stromal vascular fractions from fed Con and KO mice on RD ( n = 3 ) . Values are mean ± s . e . m . P values are as compared with diet ‐ and age ‐ matched controls . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . Con , control ; eWAT , epididymal fat ; FACS , fluorescence ‐ activated cell sorting ; HDF , high ‐ fat diet ; Iso , isoproterenol ; KO , knockout ; POMC , proopiomelanocortin ; RD , regular chow ; Stv , fasted .

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

(H) Immunoblot for oxidized proteins in eWAT from RD‐fed Con and KO mice (n=3). (I) FACS analysis for M1 and M2 macrophage proportions in adipose stromal vascular fractions from fed Con and KO mice on RD (n=3). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Con, control; eWAT, epididymal fat; FACS, fluorescence‐activated cell sorting; HDF, high‐fat diet; Iso, isoproterenol; KO, knockout; POMC, proopiomelanocortin; RD, regular chow; Stv, fasted.


( A ) Blood glucose levels in 6 mo ( n = 7 - 9 ) and ( B ) 12 mo Con and KO mice on RD ( n = 6 ) , and in ( C ) 12 ‐ mo ‐ old Con and KO mice on HFD ( n = 4 ) .

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(A) Blood glucose levels in 6 mo (n=7-9) and (B) 12 mo Con and KO mice on RD (n=6), and in (C) 12‐mo‐old Con and KO mice on HFD (n=4).


( D ) Serum insulin levels in 6 mo , ( n = 7 - 9 ) and ( E ) 12 ‐ mo ‐ old RD ‐ fed Con and KO mice that were fed or 6 h fasted ( n = 6 ) .

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(D) Serum insulin levels in 6 mo, (n=7-9) and (E) 12‐mo‐old RD‐fed Con and KO mice that were fed or 6 h fasted (n=6).


( F ) HOMA values from 6 ‐ h ‐ fasted 6 ‐ mo ‐ old ( n = 7 - 9 ) and ( G ) 12 ‐ mo ‐ old Con and KO mice ( n = 6 ) on RD . ( H ) Glucose tolerance tests in 6 ‐ h ‐ fasted 6 ‐ mo ‐ old mice on RD ( n = 4 ) and in ( I ) 6 ‐ h ‐ fasted 12 ‐ mo ‐ old Con and KO mice fed HFD for 10 mo ( n = 4 ) .

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(F) HOMA values from 6‐h‐fasted 6‐mo‐old (n=7-9) and (G) 12‐mo‐old Con and KO mice (n=6) on RD. (H) Glucose tolerance tests in 6‐h‐fasted 6‐mo‐old mice on RD (n=4) and in (I) 6‐h‐fasted 12‐mo‐old Con and KO mice fed HFD for 10 mo (n=4).


( J ) Insulin tolerance tests in RD ‐ fed 6 ‐ mo ‐ old ( n = 4 ) and ( K ) HFD ‐ fed 12 ‐ mo ‐ old Con and KO mice ( n = 4 ) .

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

(J) Insulin tolerance tests in RD‐fed 6‐mo‐old (n=4) and (K) HFD‐fed 12‐mo‐old Con and KO mice (n=4).


( L ) Insulin tolerance tests and ( M ) body weights of 1 ‐ mo ‐ old Con and KO mice on RD ( n = 3 - 5 ) . Values are mean ± s . e . m . P values are as compared with diet ‐ and age ‐ matched controls . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . Con , control ; HDF , high ‐ fat diet ; HOMA , homeostasis model of insulin resistance ; KO , knockout ; mo , month ; POMC , proopiomelanocortin ; RD , regular chow ; Stv , fasted .

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

(L) Insulin tolerance tests and (M) body weights of 1‐mo‐old Con and KO mice on RD (n=3-5). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Con, control; HDF, high‐fat diet; HOMA, homeostasis model of insulin resistance; KO, knockout; mo, month; POMC, proopiomelanocortin; RD, regular chow; Stv, fasted.


( A ) Immunoblots for Atg7 in MBH from young ( 3 mo ) , middle ‐ aged ( 12 mo ) and aged ( 22 mo ) fed and 6 ‐ h ‐ fasted ( Stv ) mice ( n = 4 ) , and ( B ) LC3 ‐ II in MBH explants from 3 mo and 22 mo fed mice in presence or absence of lysosomal inhibitors ( Inh ) ( n = 11 - 13 ) . Mean ± s . e . m values for ( B ) steady ‐ state LC3 ‐ II and ( C ) LC3 ‐ II flux are shown ( n = 11 - 13 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblots for Atg7 in MBH from young (3 mo), middle‐aged (12 mo) and aged (22 mo) fed and 6‐h‐fasted (Stv) mice (n=4), and (B) LC3‐II in MBH explants from 3 mo and 22 mo fed mice in presence or absence of lysosomal inhibitors (Inh) (n=11-13). Mean±s.e.m values for (B) steady‐state LC3‐II and (C) LC3‐II flux are shown (n=11-13).


( D ) Immunoblots for p62 and NBR1 flux in MBH from 3 mo and 22 mo fed mice in presence or absence of Inh ( n = 11 - 13 ) .

O O O O O O O O O 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) Immunoblots for p62 and NBR1 flux in MBH from 3 mo and 22 mo fed mice in presence or absence of Inh (n=11-13).


( E ) Immunofluorescence microscopy for POMC ( green ) and p62 ( red ) ( n = 4 ) , ( F ) immunoblots for POMC and ACTH ( n = 4 ) , and ( G ) immunofluorescence for α ‐ MSH in MBH from 3 mo and 22 mo mice on RD ( n = 4 ) .

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

(E) Immunofluorescence microscopy for POMC (green) and p62 (red) (n=4), (F) immunoblots for POMC and ACTH (n=4), and (G) immunofluorescence for α‐MSH in MBH from 3 mo and 22 mo mice on RD (n=4).


( H ) Body wt and ( I ) eWAT wt of 3 ‐ mo ‐ and 22 ‐ mo ‐ old mice on RD ( n = 9 - 13 ) .

(H) Body wt and (I) eWAT wt of 3‐mo‐ and 22‐mo‐old mice on RD (n=9-13).


( J ) Serum fatty acids and ( K ) glycerol from fed or 6 ‐ h ‐ fasted ( Stv ) 3 mo and 22 mo mice ( n = 7 - 9 ) , and ( L , M ) after intraperitoneal Iso in 6 ‐ h ‐ fasted 3 mo and 22 mo mice ( n = 5 - 6 ) .

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

(J) Serum fatty acids and (K) glycerol from fed or 6‐h‐fasted (Stv) 3 mo and 22 mo mice (n=7-9), and (L,M) after intraperitoneal Iso in 6‐h‐fasted 3 mo and 22 mo mice (n=5-6).


( N ) Immunoblots for adipose triglyceride lipase ( ATGL ) in eWAT from 3 mo and 22 mo mice . Values are mean ± s . e . m . P values are as compared with controls . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 . Nuclei are blue ( DAPI ) . Scale inset : 10 μm . Yellow arrows indicate merged signal . ACTH , adrenocorticotrophic hormone ; Con , control ; eWAT , epididymal fat ; Iso , isoproterenol ; MBH , mediobasal hypothalamic ; mo , months ; MSH , melanocyte ‐ stimulating hormone ; POMC , proopiomelanocortin ; RD , regular chow ; wt , weight .

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

(N) Immunoblots for adipose triglyceride lipase (ATGL) in eWAT from 3 mo and 22 mo mice. Values are mean±s.e.m. P values are as compared with controls. *P0.05, **P0.01, ***P0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. Yellow arrows indicate merged signal. ACTH, adrenocorticotrophic hormone; Con, control; eWAT, epididymal fat; Iso, isoproterenol; MBH, mediobasal hypothalamic; mo, months; MSH, melanocyte‐stimulating hormone; POMC, proopiomelanocortin; RD, regular chow; wt, weight.


( B ) Weight gain and ( C ) area under the curve of percentage weight gain of HFD - fed mice compared to RD - fed mice , treated with vehicle or antibiotic ; n = 13 ( RD ) , 17 ( HFD ) , 16 ( HFD + AB ) , 12 ( RD + AB ) ; CI 99 . 9 %

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Weight gain and (C) area under the curve of percentage weight gain of HFD-fed mice compared to RD-fed mice, treated with vehicle or antibiotic; n= 13(RD), 17(HFD), 16(HFD+AB), 12(RD+AB); CI 99.9%


( D ) Compilation of compressed Z - stack confocal image of FITC - dextran - labeled CNV and single - plane confocal image of isolectin B4 - stained choroidal flat mounts from RD - and HFD - fed mice , with vehicle or antibiotic treatment . ( E ) Quantification of area of FITC - dextran - labeled CNV over isolectin B4 - stained laser impact area fold RD ; n = 11 ( RD ) , 16 ( HFD ) , 11 ( HFD + AB ) , 12 ( RD + AB ) , with 36 ( RD ) , 32 ( HFD ) , 33 ( HFD + AB ) , 34 ( RD + AB ) burns total ; CI 99 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Compilation of compressed Z-stack confocal image of FITC-dextran-labeled CNV and single-plane confocal image of isolectin B4-stained choroidal flat mounts from RD- and HFD-fed mice, with vehicle or antibiotic treatment. (E) Quantification of area of FITC-dextran-labeled CNV over isolectin B4-stained laser impact area fold RD; n= 11(RD), 16(HFD), 11(HFD+AB), 12(RD+AB), with 36(RD), 32(HFD), 33(HFD+AB), 34(RD+AB) burns total; CI 99%.


( F ) Representative circle charts of relative abundance of bacterial phyla , class , order and family ( from central to peripheral ) , in gut microbiota of RD - fed mice with vehicle , HFD - fed mice with , HFD - fed mice with neomycin and RD - fed mice with neomycin and ( G ) relative proportion per group of different phyla ; n = 7 ( RD ) , 8 ( HFD ) , 6 ( HFD + AB ) , 7 ( RD + AB ) ; Bacteroidetes ; CI 99 . 9 % , Firmicutes ; CI 99 % , Proteobacteria ; CI 99 % , Actinobacteria and Spirochaetes .

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

(F) Representative circle charts of relative abundance of bacterial phyla, class, order and family (from central to peripheral), in gut microbiota of RD-fed mice with vehicle, HFD-fed mice with, HFD-fed mice with neomycin and RD-fed mice with neomycin and (G) relative proportion per group of different phyla; n= 7(RD), 8(HFD), 6(HFD+AB), 7(RD+AB); Bacteroidetes; CI 99.9%, Firmicutes; CI 99%, Proteobacteria; CI 99%, Actinobacteria and Spirochaetes.


( A - C ) 3D rendering of Z - stack confocal image showing Isolectin B4 - stained laser - burn with FITC - dextran - labeled CNV and IBA - 1 - stained MPs .

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

(A-C) 3D rendering of Z-stack confocal image showing Isolectin B4-stained laser-burn with FITC-dextran-labeled CNV and IBA-1-stained MPs.


( D ) Representative FACS plots of retinas and sclera - choroid - RPE - cell complexes from Regular - chow Diet ( RD ) - fed mice without and 3 , 7 and 14 days after laser - burn . ( E ) Quantification of MPs ( Ly6G - , F4 / 80 + , CD11b + ) at p3 , p7 and p14 ; fold naïve ( no burn ) ; n = 5 ( no burn ) , 5 ( p3 ) , 6 ( p7 ) , 5 ( p14 ) ; CI 95 % . ( F ) Representative FACS plots and ( G ) quantification of microglia ( Ly6G - , F4 / 80 + , CD11b + , CX3CR1hiCD45lo ) fold naïve ; n = 5 ( no burn ) , 4 ( p3 ) , 6 ( p7 ) , 5 ( p14 ) ; CI 95 % .

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

(D) Representative FACS plots of retinas and sclera-choroid-RPE-cell complexes from Regular-chow Diet (RD)-fed mice without and 3, 7 and 14 days after laser-burn. (E) Quantification of MPs (Ly6G -, F4/80+, CD11b+) at p3, p7 and p14; fold naïve (no burn); n= 5(no burn), 5(p3), 6(p7), 5(p14); CI 95%. (F) Representative FACS plots and (G) quantification of microglia (Ly6G-, F4/80+, CD11b+, CX3CR1hiCD45lo) fold naïve; n= 5(no burn), 4(p3), 6(p7), 5(p14); CI 95%.


( H ) Representative FACS plots of retinas and sclera - choroid - RPE - cell complexes from RD - and HFD - fed mice , with vehicle or antibiotic treatment at p7 . ( I ) Quantification of MPs at p7 fold RD ; n = 6 ( RD ) , 4 ( HFD ) , 3 ( HFD + AB ) , 4 ( RD + AB ) ; CI 95 % . ( J ) Representative FACS plots and ( K ) quantification of microglia ( Ly6G - , F4 / 80 + , CD11b + , CX3CR1hiCD45lo ) fold RD ; n = 5 ( RD ) , 5 ( HFD ) , 3 ( HFD + AB ) , 4 ( RD + AB ) ; CI 95 % .

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

(H) Representative FACS plots of retinas and sclera-choroid-RPE-cell complexes from RD- and HFD-fed mice, with vehicle or antibiotic treatment at p7. (I) Quantification of MPs at p7 fold RD; n= 6(RD), 4(HFD), 3(HFD+AB), 4(RD+AB); CI 95%. (J) Representative FACS plots and (K) quantification of microglia (Ly6G-, F4/80+, CD11b+, CX3CR1hiCD45lo) fold RD; n= 5(RD), 5(HFD), 3(HFD+AB), 4(RD+AB); CI 95%.


( L ) Representative confocal images of IBA - 1 - stained MPs on choroidal flat mounts from RD - and HFD - fed mice , treated with vehicle or with neomycin . Examples of labeled macrophages ( white dots ) are presented in side - panels . ( M ) Total number of MPs around laser impact area ; n = 7 ( RD ) , 9 ( HFD ) , 4 ( HFD + AB ) , 7 ( RD + AB ) , with 23 ( RD ) , 29 ( HFD ) , 13 ( HFD + AB ) , 23 ( RD + AB ) burns total ; CI 99 % .

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

(L) Representative confocal images of IBA-1-stained MPs on choroidal flat mounts from RD- and HFD-fed mice, treated with vehicle or with neomycin. Examples of labeled macrophages (white dots) are presented in side-panels. (M) Total number of MPs around laser impact area; n= 7(RD), 9(HFD), 4(HFD+AB), 7(RD+AB), with 23(RD), 29(HFD), 13(HFD+AB), 23(RD+AB) burns total; CI 99%.


( A ) Concentration of gut absorbed Evans Blue in serum 24 hours after oral administration in Regular - chow Diet ( RD ) - and high - fat diet ( HFD ) - fed mice , treated with vehicle or with antibiotic treatment ; n = 6 ( RD ) , 5 ( HFD ) , 5 ( HFD + AB ) , 5 ( RD + AB ) ; CI 99 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Concentration of gut absorbed Evans Blue in serum 24 hours after oral administration in Regular-chow Diet (RD)- and high-fat diet (HFD)-fed mice, treated with vehicle or with antibiotic treatment; n= 6(RD), 5(HFD), 5(HFD+AB), 5(RD+AB); CI 99%.


( B ) Activation of PRRs induced by stimulation with serum isolated from mice fed RD or HFD for 7 weeks and receiving vehicle or neomycin for 3 weeks ; fold RD ; n = 3 for all groups ; CI 99 % .

O 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

(B) Activation of PRRs induced by stimulation with serum isolated from mice fed RD or HFD for 7 weeks and receiving vehicle or neomycin for 3 weeks; fold RD; n=3 for all groups; CI 99%.


( C ) Serum cytokine profiles determined by Bio - Plex assay ( Bio - Rad ) of IL - 1β , n = 11 ( RD ) , 12 ( HFD ) , 12 ( HFD + AB ) , 11 ( RD + AB ) ; CI 95 % ; IL - 6 , n = 11 ( RD ) , 12 ( HFD ) , 12 ( HFD + AB ) , 11 ( RD + AB ) , CI 99 . 9 % ; TNF - α n = 11 for all groups , CI 99 % ; IL - 17A n = 11 ( RD ) , 12 ( HFD ) , 12 ( HFD + AB ) , 11 ( RD + AB ) ; CI 95 % ; and IFN - γ ; n = 12 for all groups ; CI 95 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Serum cytokine profiles determined by Bio-Plex assay (Bio-Rad) of IL-1β, n= 11(RD), 12(HFD), 12(HFD+AB), 11(RD+AB); CI 95%; IL-6, n= 11(RD), 12(HFD), 12(HFD+AB), 11(RD+AB), CI 99.9%; TNF-α n=11 for all groups, CI 99%; IL-17A n= 11(RD), 12(HFD), 12(HFD+AB), 11(RD+AB); CI 95%; and IFN-γ; n= 12 for all groups; CI 95%.


( D ) mRNA expression of Il - 1β ; n = 5 ( RD ) , 7 ( HFD ) , 7 ( HFD + AB ) , 3 ( RD + AB ) ; CI 95 % , Il - 6 ; n = 5 ( RD ) , 5 ( HFD ) , 4 ( HFD + AB ) , 4 ( RD + AB ) ; CI 95 % , Tnf - α ; n = 6 ( RD ) , 9 ( HFD ) , 4 ( HFD + AB ) , 5 ( RD + AB ) ; CI 95 % and Vegf - a ; n = 6 ( RD ) , 5 ( HFD ) , 5 ( HFD + AB ) , 6 ( RD + AB ) ; CI 95 % in choroids .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) mRNA expression of Il-1β; n= 5(RD), 7(HFD), 7(HFD+AB), 3(RD+AB); CI 95%, Il-6; n= 5(RD), 5(HFD), 4(HFD+AB), 4(RD+AB); CI 95%, Tnf-α; n= 6(RD), 9(HFD), 4(HFD+AB), 5(RD+AB); CI 95% and Vegf-a; n= 6(RD), 5(HFD), 5(HFD+AB), 6(RD+AB); CI 95% in choroids.


( C ) Weight gain and ( D ) area under the curve of percentage weight gain of RD x RDT , HFD x RDT and HFD x HFDT mice ; n = 13 ( RDxRDT ) , 12 ( HFDxHFDT ) , 14 ( HFDxRDT ) ; CI 99 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Weight gain and (D) area under the curve of percentage weight gain of RD x RDT, HFD x RDT and HFD x HFDT mice; n= 13(RDxRDT), 12(HFDxHFDT), 14(HFDxRDT); CI 99%.


( E ) Representative circle charts of relative abundance of bacterial phyla in gut microbiota of RD - fed mice receiving a RD transfer and HFD - fed mice receiving a HFD or RD

O O O O O O O O O O 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) Representative circle charts of relative abundance of bacterial phyla in gut microbiota of RD-fed mice receiving a RD transfer and HFD-fed mice receiving a HFD or RD transfer


( F ) relative proportion per experimental group of different phyla ; n = 5 ( RDxRDT ) , 5 ( HFDxHFDT ) , 4 ( HFDxRDT ) ; Bacteroidetes ; CI 99 % , Firmicutes ; CI 95 % , Proteobacteria ; CI 95 % , Actinobacteria and Spirochaetes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) relative proportion per experimental group of different phyla; n= 5(RDxRDT), 5(HFDxHFDT), 4(HFDxRDT); Bacteroidetes; CI 99%, Firmicutes; CI 95%, Proteobacteria; CI 95%, Actinobacteria and Spirochaetes.


( G ) Compilation of compressed Z - stack confocal images of FITC - dextran - labeled CNV and single - plane confocal images of isolectin B4 - stained choroidal flat mounts from RD x RDT , HFD x RDT and HFD x HFDT mice . ( H ) Quantification of area of FITC - dextran - labeled CNV over area of isolectin B4 - stained laser impact area fold RD x RDT ; n = 6 ( RDxRDT ) , 9 ( HFDxHFDT ) , 9 ( HFDxRDT ) , with 15 ( RDxRDT ) , 21 ( HFDxHFDT ) , 16 ( HFDxRDT ) burns total ; CI 95 % .

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

(G) Compilation of compressed Z-stack confocal images of FITC-dextran-labeled CNV and single-plane confocal images of isolectin B4-stained choroidal flat mounts from RD x RDT, HFD x RDT and HFD x HFDT mice. (H) Quantification of area of FITC-dextran-labeled CNV over area of isolectin B4-stained laser impact area fold RD x RDT; n= 6(RDxRDT), 9(HFDxHFDT), 9(HFDxRDT), with 15(RDxRDT), 21(HFDxHFDT), 16(HFDxRDT) burns total; CI 95%.


( I ) Concentration of gut absorbed Evans Blue in serum 24 hours after oral administration in RD - and HFD - fed mice , after RD or HFD microbiotal transplant ; n = 3 ( RDxRDT ) , 3 ( HFDxHFDT ) , 4 ( HFDxRDT ) ; CI 95 % .

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

(I) Concentration of gut absorbed Evans Blue in serum 24 hours after oral administration in RD- and HFD-fed mice, after RD or HFD microbiotal transplant; n= 3(RDxRDT), 3(HFDxHFDT), 4(HFDxRDT); CI 95%.


( J ) mRNA expression of Il - 1β ; n = 5 ( RDxRDT ) , 4 ( HFDxHFDT ) , 5 ( HFDxRDT ) , Il - 6 ; n = 5 ( RDxRDT ) , 4 ( HFDxHFDT ) , 4 ( HFDxRDT ) ; CI 99 % , Tnf - α ; n = 5 ( RDxRDT ) , 4 ( HFDxHFDT ) , 4 ( HFDxRDT ) ; CI 95 % and Vegf - a ; n = 5 ( RDxRDT ) , 4 ( HFDxHFDT ) , 5 ( HFDxRDT ) ; CI 95 % in choroids .

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

(J) mRNA expression of Il-1β; n= 5(RDxRDT), 4(HFDxHFDT), 5(HFDxRDT), Il-6; n= 5(RDxRDT), 4(HFDxHFDT), 4(HFDxRDT); CI 99%, Tnf-α; n= 5(RDxRDT), 4(HFDxHFDT), 4(HFDxRDT); CI 95% and Vegf-a; n= 5(RDxRDT), 4(HFDxHFDT), 5(HFDxRDT); CI 95% in choroids.


( A ) PKH26 ( PE ) - labelled RM1 ( V500 / eCFP ) cells were injected intracardiac ( IC ) into C57BL / 6 mice and FACS - isolated from bones with evident tumor burden ( 8 individual mice across 5 independent experiments ) from day 16 onward and individual dormant and proliferating cells were isolated for scRNA - seq . Representative tumor burden at the whole mouse level and in bone shown by bioluminescence with representative FACS plots of PKH + ( PE / V500 ) and PKH - ( V500 ) RM1 cells and bone marrow cells ( grey ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PKH26 (PE)-labelled RM1 (V500/eCFP) cells were injected intracardiac (IC) into C57BL/6 mice and FACS-isolated from bones with evident tumor burden (8 individual mice across 5 independent experiments) from day 16 onward and individual dormant and proliferating cells were isolated for scRNA-seq. Representative tumor burden at the whole mouse level and in bone shown by bioluminescence with representative FACS plots of PKH+ (PE/V500) and PKH- (V500) RM1 cells and bone marrow cells (grey).


( B ) goana gene ontology ( GO ) analysis ( limma ) of all DE genes enriched in proliferating ( PKH - ; n = 32 ) cells compared to dormant cells ( PKH + ; n = 28 ) . Gene sets appear in order of significance ( p value ) with color representing fold enrichment and bar width indicating the number of genes in each process .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) goana gene ontology (GO) analysis (limma) of all DE genes enriched in proliferating (PKH-; n = 32) cells compared to dormant cells (PKH+; n = 28). Gene sets appear in order of significance (p value) with color representing fold enrichment and bar width indicating the number of genes in each process.


( C ) goana GO analysis ( limma ) of all DE genes uniquely enriched in PKH + compared to PKH - cells . Gene sets appear in order of significance ( p value ) with color representing fold enrichment and bar width indicating the number of genes in each process .

(C) goana GO analysis (limma) of all DE genes uniquely enriched in PKH+ compared to PKH- cells. Gene sets appear in order of significance (p value) with color representing fold enrichment and bar width indicating the number of genes in each process.


( E ) Heatmap of relative BASiCS - derived log2 ( denoised counts + 1 ) of type I IRGs ( upregulated > 2 - fold in the INTERFEROME in at least one dataset ) that are differentially expressed between all dormant and active cells with no residual over - dispersion . Mice IDs for each sample are indicated , along with type I IRGs that are identified as such in at least 30 INTERFEROME datasets . Genes are displayed if detected in at least 10 samples per population . Samples with zero counts for individual genes are represented in white .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmap of relative BASiCS-derived log2(denoised counts + 1) of type I IRGs (upregulated > 2-fold in the INTERFEROME in at least one dataset) that are differentially expressed between all dormant and active cells with no residual over-dispersion. Mice IDs for each sample are indicated, along with type I IRGs that are identified as such in at least 30 INTERFEROME datasets. Genes are displayed if detected in at least 10 samples per population. Samples with zero counts for individual genes are represented in white.


( F ) Dot plot of BASiCS - derived log2 ( denoised counts + 1 ) of key DE IRGs expressed in > 5 dormant cells associated with immune - activatory processes ( among one of the most enriched types of biological pathway determined by GO analyses ) present for all single cells ranked by ExpLogFC ( Fig . 1c ) . Open circles are zero counts . Bars indicate mean expression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dot plot of BASiCS-derived log2(denoised counts + 1) of key DE IRGs expressed in > 5 dormant cells associated with immune-activatory processes (among one of the most enriched types of biological pathway determined by GO analyses) present for all single cells ranked by ExpLogFC (Fig. 1c). Open circles are zero counts. Bars indicate mean expression.


( A Bulk RNA - seq analysis of DE genes ( DEG ) significantly suppressed ( FDR < 0 . 05 ; generalized linear model extraction ( GLM ) by edgeR ) in proliferating RM1 bone - derived ( BD ) cells compared to parental RM1 and lung metastases . Tumor - intrinsic type I IFN signaling is suppressed in proliferating bone lesions ( n = 1 ) compared to parental RM1 ( n = 2 ) cells as shown by ( A ) camera analysis of Hallmark gene set responses associated with bone metastasis ( bar length is the - log10FDR ; false discovery rate ) . Color indicates mean log2fold change ( FC ) of all genes in gene set . Bar width is relative gene set size . Dashed line shows FDR = 0 . 05 ; ( B ) barcode plot showing enrichment of the Hallmark IFN - α response gene set in genes suppressed in bone metastases , using the signed log - likelihood ratio statistic ( EdgeR ) . Bars indicate value of the statistic for each gene in the gene set ;

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(A Bulk RNA-seq analysis of DE genes (DEG) significantly suppressed (FDR < 0.05; generalized linear model extraction (GLM) by edgeR) in proliferating RM1 bone-derived (BD) cells compared to parental RM1 and lung metastases. Tumor-intrinsic type I IFN signaling is suppressed in proliferating bone lesions (n = 1) compared to parental RM1 (n = 2) cells as shown by (A) camera analysis of Hallmark gene set responses associated with bone metastasis (bar length is the -log10FDR; false discovery rate). Color indicates mean log2fold change (FC) of all genes in gene set. Bar width is relative gene set size. Dashed line shows FDR = 0.05; (B) barcode plot showing enrichment of the Hallmark IFN-α response gene set in genes suppressed in bone metastases, using the signed log-likelihood ratio statistic (EdgeR). Bars indicate value of the statistic for each gene in the gene set;


( C ) log2FC values of key downregulated IRGs , with genes retained in dormant cells and classical downstream IFN signaling targets indicated . Altered IRGs directly involved in IFN - α / β production ( light blue ) compared to downstream IFN targets ( dark blue ) are segregated . p values represented as * < 0 . 05 , * * < 0 . 005 and * * * < 0 . 0005 ; GLM by edgeR .

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(C) log2FC values of key downregulated IRGs, with genes retained in dormant cells and classical downstream IFN signaling targets indicated. Altered IRGs directly involved in IFN-α/β production (light blue) compared to downstream IFN targets (dark blue) are segregated. p values represented as * < 0.05, ** < 0.005 and *** < 0.0005; GLM by edgeR.