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 ) .

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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 .

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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 .

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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 .

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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 ) .

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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 ) .

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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 )

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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 .

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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 . )

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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 :

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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 .

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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 :

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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 .

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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 )

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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 .

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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 .

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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

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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

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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 .

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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 .

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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 .

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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 .

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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 :

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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 .

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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 .

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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 :

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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 .

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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 .

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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 .

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

(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 ) .

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

(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 B-EXP_ASSAY O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O 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 ) .

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(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 .

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(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 B-EXP_ASSAY O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD 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 ) .

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(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 ) .

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(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 ) .

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(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 ) .

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(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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 %

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(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 B-EXP_ASSAY O B-EXP_ASSAY O O B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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

(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 B-TISSUE O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

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

(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 % .

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(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

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(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 .

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(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 B-EXP_ASSAY O B-EXP_ASSAY O O B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O 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 .

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

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

(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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY 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 ;

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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) 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.


( E ) Heatmap of mean normalized voom expression of IRGs suppressed in RM1 bone metastases ( n = 1 ) compared to lung metastases ( n = 1 ) and parental cells ( n = 2 ) .

O O O O O O O O B-EXP_ASSAY O O O O B-CELL_LINE O 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 mean normalized voom expression of IRGs suppressed in RM1 bone metastases (n = 1) compared to lung metastases (n = 1) and parental cells (n = 2).


( F ) qRT - PCR validation of Irf7 and Irf9 downregulation in RM1 cells from bone metastases ( RMI BD ) compared to parental RM1 cells , lung metastases ( RM1 lung ) and naïve bone marrow ( BM ) ( n = 3 mice per group ) . p values represented as * * < 0 . 005 and * * * < 0 . 0005 ( Student ' s t test ) .

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

(F) qRT-PCR validation of Irf7 and Irf9 downregulation in RM1 cells from bone metastases (RMI BD) compared to parental RM1 cells, lung metastases (RM1 lung) and naïve bone marrow (BM) (n = 3 mice per group). p values represented as ** < 0.005 and *** < 0.0005 (Student's t test).


( G ) qRT - PCR of Irf7 and Irf9 downregulation in parental RM1 cells , RM1 cells from bone metastases ( RM1 BD ) in WT and Ifnar1 deficient ( - / - ) mice , with naïve BM from WT and Ifnar1 ( - / - ) animals for reference . ( n = 3 mice per group ; n = 1 for - / - BM control ) . p values represented as * < 0 . 05 and * * < 0 . 005 ( Student ' s t test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O B-CELL_LINE O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O 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) qRT-PCR of Irf7 and Irf9 downregulation in parental RM1 cells, RM1 cells from bone metastases (RM1 BD) in WT and Ifnar1 deficient (-/-) mice, with naïve BM from WT and Ifnar1 (-/-) animals for reference. (n = 3 mice per group; n = 1 for -/- BM control). p values represented as * < 0.05 and ** < 0.005 (Student's t test).


( A ) Stability of Irf7 and Irf9 mRNA suppression by qRT - PCR in ex vivo bone - derived cells ( RM1 BD Irf - ; n = 7 ) in culture compared to a bone - derived line that showed initial loss during early passage ( RM1 BD REV ( EP ) ; n = 3 ) then reverted to parental expression levels at late passage ( RM1 BD REV ( LP ) ; n = 4 ) compared to parental RM1 ( n = 3 ) .

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

(A) Stability of Irf7 and Irf9 mRNA suppression by qRT-PCR in ex vivo bone-derived cells (RM1 BD Irf-; n = 7) in culture compared to a bone-derived line that showed initial loss during early passage (RM1 BD REV (EP); n = 3) then reverted to parental expression levels at late passage (RM1 BD REV (LP); n = 4) compared to parental RM1 (n = 3).


( B ) ELISA of IFN - α production by RM1 parental ( n = 3 ) , RM1 bone - derived Irf low ( RM1 BD Irf - ; n = 4 ) and RM1 BD REV cells ( n = 3 ) subsequent to poly I : C stimulation .

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

(B) ELISA of IFN-α production by RM1 parental (n = 3), RM1 bone-derived Irf low (RM1 BD Irf-; n = 4) and RM1 BD REV cells (n = 3) subsequent to poly I:C stimulation.


( C ) qRT - PCR analysis of Irf7 and Irf9 expression in RM1 BD Irf - cells ± 48 h treatment with MS275 ( 1 μM ) ( n = 7 - 9 ) .

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

(C) qRT-PCR analysis of Irf7 and Irf9 expression in RM1 BD Irf- cells ± 48 h treatment with MS275 (1 μM) (n = 7-9).


( e ) qRT - PCR analysis of Irf7 and Irf9 expression in parental RM1 cells ( n = 4 ) 48 h post - contact culture with naïve BM ( n = 6 ) .

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

(e) qRT-PCR analysis of Irf7 and Irf9 expression in parental RM1 cells (n = 4) 48 h post-contact culture with naïve BM (n = 6).


( F ) qRT - PCR analysis of Irf9 expression in parental RM1 cells ± 48 h co - culture with naïve BM under contact ( non - transwell ; NT ) and transwell ( 0 . 4 μm filters that prevent cell contact ) conditions ( n = 6 - 8 per condition ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O 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) qRT-PCR analysis of Irf9 expression in parental RM1 cells ± 48 h co-culture with naïve BM under contact (non-transwell; NT) and transwell (0.4 μm filters that prevent cell contact) conditions (n = 6-8 per condition).


( G ) qRT - PCR analysis of Irf9 expression in parental RM1 cells ± 48 h contact co - culture with naïve BM ± MS275 ( 1 μM ) ( n = 3 - 6 per condition ) .

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

(G) qRT-PCR analysis of Irf9 expression in parental RM1 cells ± 48 h contact co-culture with naïve BM ± MS275 (1 μM) (n = 3-6 per condition).


( A ) Bone metastasis - free survival in WT C57BL / 6 mice harboring RM1 parental , RM1 BD Irf - and RM1 BD REV ( LP ) tumors ( n = 5 per group ; * * p = 0 . 0043 by log - rank ( Mantel - Cox ) test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O B-ORGANISM O B-CELL_LINE O O B-CELL_LINE O B-SUBCELLULAR O O B-CELL_LINE O O O O O O O O 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) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 parental, RM1 BD Irf- and RM1 BD REV (LP) tumors (n = 5 per group; **p = 0.0043 by log-rank (Mantel-Cox) test).


( C ) ELISA of IFN - α production by RM1 parental , RM1 BD Irf - base vector ( BV ) and RM1 BD Irf7 over - expressing ( OE ) with 24 h poly I : C stimulation ( n = 4 ) .

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

(C) ELISA of IFN-α production by RM1 parental, RM1 BD Irf- base vector (BV) and RM1 BD Irf7 over-expressing (OE) with 24 h poly I:C stimulation (n = 4).


( D ) Bone metastasis - free survival in WT C57BL / 6 mice subsequent to IC inoculation of RM1 BD Irf - BV ( n = 9 ) and RM1 BD Irf7 OE ( n = 7 ) cells ( * * p = 0 . 0028 by log - rank ( Mantel - Cox ) test ) .

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

(D) Bone metastasis-free survival in WT C57BL/6 mice subsequent to IC inoculation of RM1 BD Irf- BV (n = 9) and RM1 BD Irf7 OE (n = 7) cells (**p = 0.0028 by log-rank (Mantel-Cox) test).


( E ) Bone metastasis - free survival in Ifnar1 - / - mice harboring RM1 BD Irf - BV and RM1 BD Irf7 OE tumors ( n = 4 per group ) and representative bioluminescent imaging of tumor burden in leg bones ( also shown in Appendix Fig S4F ) .

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

(E) Bone metastasis-free survival in Ifnar1 -/- mice harboring RM1 BD Irf- BV and RM1 BD Irf7 OE tumors (n = 4 per group) and representative bioluminescent imaging of tumor burden in leg bones (also shown in Appendix Fig S4F).


( F ) Immunohistochemical ( IHC ) staining for cerulean ( anti - eCFP ; green ) on naïve WT bone , RM1 parental , RM1 BD Irf - BV and RM1 BD Irf7 OE tumor - bearing bones derived from WT animals ; and RM1 BD Irf7 OE tumor - bearing bones derived from Ifnar1 - / - animals at survival assay endpoints . Blue represents DAPI nuclear staining . Scale bar , 100 μm .

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

(F) Immunohistochemical (IHC) staining for cerulean (anti-eCFP; green) on naïve WT bone, RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE tumor-bearing bones derived from WT animals; and RM1 BD Irf7 OE tumor-bearing bones derived from Ifnar1 -/- animals at survival assay endpoints. Blue represents DAPI nuclear staining. Scale bar, 100 μm.


( G - I ) FACS analysis of dormant ( claret + ) RM1 BD Irf - and RM1 BD Irf7 OE cells from bone at ( G ) day 11 post - IC injection ( n = 4 per group ) and ( h ) day 17 ( n = 4 - 6 mice group / time point ) with ( I ) D17 active ( claret - ) colony quantitation ( mean + 1 ) determined by multiphoton imaging and IMARIS interrogation ( n = 9 bones from 3 mice per condition ) . Median shown . Upper and low box hinges denote first and third quartiles . Whiskers mark value limits .

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

(G-I) FACS analysis of dormant (claret+) RM1 BD Irf- and RM1 BD Irf7 OE cells from bone at (G) day 11 post-IC injection (n = 4 per group) and (h) day 17 (n = 4-6 mice group/time point) with (I) D17 active (claret-) colony quantitation (mean +1) determined by multiphoton imaging and IMARIS interrogation (n = 9 bones from 3 mice per condition). Median shown. Upper and low box hinges denote first and third quartiles. Whiskers mark value limits.


( J ) FACS analysis of IFN - γ + and TNF - α + CD8 + T cells ( % ) post - ICS induction of T cell ( spleen - derived from tumor - bearing mice ) activation by RM1 parental , RM1 BD Irf - BV and RM1 BD Irf7 OE cells ( n = 6 per condition / group ) .

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

(J) FACS analysis of IFN-γ+ and TNF-α+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from tumor-bearing mice) activation by RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE cells (n = 6 per condition/group).


( K ) Mean quantitation of TRAP - stained osteoclasts differentiated with M - CSF ± RANKL in co - culture with RM1 BD Irf - BV and RM1 BD Irf7 OE cells with representative wells shown ( n = 3 per condition M - CSF ; n = 6 per condition M - CSF + RANKL ; also shown in Appendix Fig S4J ) . Scale bar represents 100 μm .

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

(K) Mean quantitation of TRAP-stained osteoclasts differentiated with M-CSF ± RANKL in co-culture with RM1 BD Irf- BV and RM1 BD Irf7 OE cells with representative wells shown (n = 3 per condition M-CSF; n = 6 per condition M-CSF + RANKL; also shown in Appendix Fig S4J). Scale bar represents 100 μm.


( A ) ELISA of IFN - α production by RM1 BD Irf - cells ± single agent MS275 and poly I : C treatment or 48 h pre - treatment with MS275 ( pMS ) prior to poly I : C stimulation ( n = 6 ) .

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

(A) ELISA of IFN-α production by RM1 BD Irf- cells ± single agent MS275 and poly I:C treatment or 48 h pre-treatment with MS275 (pMS) prior to poly I:C stimulation (n = 6).


( B ) Mean fluorescence intensity of H2 - Kb staining on RM1 BD Irf - cells by FACS ± MS275 , poly I : C or combination treatment ( n = 3 ) .

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

(B) Mean fluorescence intensity of H2-Kb staining on RM1 BD Irf- cells by FACS ± MS275, poly I:C or combination treatment (n = 3).


( C ) FACS analysis of IFN - γ + CD8 + T cells ( % ) post - ICS induction of T cell ( spleen - derived from RM1 BD Irf - tumor - bearing mice ) activation upon re - stimulation with RM1 cells and MS275 , poly I : C or combination treatment , with NAC ( no antigen - presenting cells ) and RM1 BD Irf7 OE controls ( n = 3 ; Irf7 OE control ( C ) , n = 1 )

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

(C) FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1)


FACS analysis of IFN - γ + CD8 + T cells ( % ) post - ICS induction of T cell ( spleen - derived from RM1 BD Irf - tumor - bearing mice ) activation upon re - stimulation with RM1 cells and MS275 , poly I : C or combination treatment , with NAC ( no antigen - presenting cells ) and RM1 BD Irf7 OE controls ( n = 3 ; Irf7 OE control ( C ) , n = 1 ) with ( D ) representative FACS plots shown .

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

FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1) with (D) representative FACS plots shown.


( E ) Bone metastasis - free survival in WT C57BL / 6 mice harboring RM1 BD Irf - cells ± MS275 , poly I : C or combination treatment ( n = 6 per group ; log - rank ( Mantel - Cox ) test ) .

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

(E) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 BD Irf- cells ± MS275, poly I:C or combination treatment (n = 6 per group; log-rank (Mantel-Cox) test).


( F ) Mean total tumor burden in bone ( femurs , tibias , spine and humerus ) at endpoint by bioluminescent intensity score ( log2 p / sec / cm2 / sr ) with values < 4 . 0x104 representing zero burden ( n = 24 bones per group ; 6 mice per group ) and representative bioluminescent imaging of tumor burden in spine ( all shown in Appendix Figure S5E ) .

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

(F) Mean total tumor burden in bone (femurs, tibias, spine and humerus) at endpoint by bioluminescent intensity score (log2 p/sec/cm2/sr) with values < 4.0x104 representing zero burden (n = 24 bones per group; 6 mice per group) and representative bioluminescent imaging of tumor burden in spine (all shown in Appendix Figure S5E).


( A , FACS analysis of peripheral blood ( PB ) T and NK lymphocyte representation and activation

O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O B-EXP_ASSAY

(A, FACS analysis of peripheral blood (PB) T and NK lymphocyte representation and activation status


B ) FACS analysis of FoxP3 + CD4 + and CD4 + effector memory T cell status at days 4 and 10 post - IC tumor cell inoculation across treatment settings ( n = 4 - 6 per group ) .

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

B) FACS analysis of FoxP3+ CD4+ and CD4+ effector memory T cell status at days 4 and 10 post-IC tumor cell inoculation across treatment settings (n = 4-6 per group).


( C , Bioluminescent imaging ( C ) of a combination group mouse with tumor clearance from day 7 to 41 ( full experimental cohort shown in Appendix Fig S5E )

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

(C, Bioluminescent imaging (C) of a combination group mouse with tumor clearance from day 7 to 41 (full experimental cohort shown in Appendix Fig S5E)


D ) post - IC inoculation with associated specific CD8 + memory T cell response . This is represented by FACS analysis of IFN - γ + CD8 + T cells ( % ) post - ICS induction of T cell ( spleen - derived from tumor - bearing mice ) activation upon re - stimulation with RM1 BD Irf - cells and MS275 , poly I : C or combination treatments in vitro , with NAC and RM1 BD Irf7 OE controls ( n = 3 ; Irf7 OE control ( C ) , n = 1 ) . Representative FACS plots shown on right .

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D) post-IC inoculation with associated specific CD8+ memory T cell response. This is represented by FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from tumor-bearing mice) activation upon re-stimulation with RM1 BD Irf- cells and MS275, poly I:C or combination treatments in vitro, with NAC and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1). Representative FACS plots shown on right.


Compositional analysis of RNA - seq data ( FDR < 0 . 05 ; GLM by edgeR ) from bone metastases ( n = 9 ) and primary tumors ( n = 12 ) revealed ( A ) immune cells and ( B ) tumor cells were relative ( % of total ) across all samples , with a mean tumor purity > 85 % as determined by ARMET . Median shown . Upper and low box hinges denote first and third quartiles . Upper and lower whiskers mark the values ± 1 . 5IQR from the hinge . Data points beyond the whiskers are shown individually . p values not significant by Wilcoxon rank sum test ( R ) .

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Compositional analysis of RNA-seq data (FDR < 0.05; GLM by edgeR) from bone metastases (n = 9) and primary tumors (n = 12) revealed (A) immune cells and (B) tumor cells were relative (% of total) across all samples, with a mean tumor purity >85% as determined by ARMET. Median shown. Upper and low box hinges denote first and third quartiles. Upper and lower whiskers mark the values ± 1.5IQR from the hinge. Data points beyond the whiskers are shown individually. p values not significant by Wilcoxon rank sum test (R).


Hallmark gene set responses associated with bone metastasis by camera analysis shows ( C ) enrichment of type I ( IFN - α ) IFN responses in suppressed genes ( barcode plot ) .

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Hallmark gene set responses associated with bone metastasis by camera analysis shows (C) enrichment of type I (IFN-α) IFN responses in suppressed genes (barcode plot).


( D ) Heatmap of type I IFN DE IRGs ( normalized log2CPM ; counts per million ) significantly suppressed in bone metastases ( n = 9 ) compared to primary ( n = 12 ) and benign ( n = 3 ) tumors , grouped by type I IFN specific , and type II and III IFN co - regulation status . Scale truncated at ± 5 relative log2CPM . GLM by edgeR .

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(D) Heatmap of type I IFN DE IRGs (normalized log2CPM; counts per million) significantly suppressed in bone metastases (n = 9) compared to primary (n = 12) and benign (n = 3) tumors, grouped by type I IFN specific, and type II and III IFN co-regulation status. Scale truncated at ± 5 relative log2CPM. GLM by edgeR.


( E ) IHC for IRF9 expression in primary prostate tumors and bone metastases with matched samples indicated . IRF9 indicated by brown ( DAB ) staining . Scale bar represents 100 μm . Full - face slides shown in Appendix Fig S7C .

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(E) IHC for IRF9 expression in primary prostate tumors and bone metastases with matched samples indicated. IRF9 indicated by brown (DAB) staining. Scale bar represents 100 μm. Full-face slides shown in Appendix Fig S7C.


( F ) Heatmap of HLA genes ( normalized log2CPM ) suppressed ( p < 0 . 05 ; FDR < 0 . 05 ) in bone metastases ( n = 9 ) , primary tumors ( n = 12 ) and benign tumors ( n = 3 ) . Scale truncated at ± 5 relative log2CPM . GLM by edgeR .

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(F) Heatmap of HLA genes (normalized log2CPM) suppressed (p <0.05; FDR <0.05) in bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). Scale truncated at ±5 relative log2CPM. GLM by edgeR.


( G ) CIBERSORT analysis of absolute leukocyte proportions ( arbitrary units , AU ) in mRNA samples of bone metastases ( n = 9 ) , primary tumors ( n = 12 ) and benign tumors ( n = 3 ) . p values by Mann - Whitney U .

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(G) CIBERSORT analysis of absolute leukocyte proportions (arbitrary units, AU) in mRNA samples of bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). p values by Mann-Whitney U.


( H ) Kaplan Meier Curve of human prostate ( n = 499 from 498 TCGA samples ) biochemical recurrence based on primary tumor alterations in a core 8 - IRG signature ( Appendix Fig S7D ) . Patient groups stratified by mRNA z - Score ( RNA - seq V2 RSEM ) ± 1 . 5 . Hazard ratio 1 . 674 ( 95 % confidence interval ( CI ) 2 . 707 to 1 . 035 ) ; * p = 0 . 03 by log - rank test .

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(H) Kaplan Meier Curve of human prostate (n = 499 from 498 TCGA samples) biochemical recurrence based on primary tumor alterations in a core 8-IRG signature (Appendix Fig S7D). Patient groups stratified by mRNA z-Score (RNA-seq V2 RSEM) ± 1.5. Hazard ratio 1.674 (95% confidence interval (CI) 2.707 to 1.035); *p = 0.03 by log-rank test.


Viability of single and double mutants of RNAPII and rad52Δ , mre11Δ and cdc44 ‐ 8 . Tenfold serial dilutions of double mutants obtained by genetic crosses were tested for growth in plain SC medium or supplemented with 10 mM HU or 0 . 005 % MMS .

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Viability of single and double mutants of RNAPII and rad52Δ, mre11Δ and cdc44‐8. Tenfold serial dilutions of double mutants obtained by genetic crosses were tested for growth in plain SC medium or supplemented with 10 mM HU or 0.005% MMS.


Tetrad analysis of rpb1 ‐ S751F , rad52Δ and mre11Δ crosses . Δ indicates double mutants that fail to grow .

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Tetrad analysis of rpb1‐S751F, rad52Δ and mre11Δ crosses. Δ indicates double mutants that fail to grow.


Tetrad analysis of rpb1 ‐ S751F crossed by pol32Δ and rad51Δ . Δ indicates triple mutants that fail to grow .

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Tetrad analysis of rpb1‐S751F crossed by pol32Δ and rad51Δ. Δ indicates triple mutants that fail to grow.


HU and MMS sensitivity of different mutant combinations of rpb1 ‐ 1 , rpb1 ‐ S751F , pol32Δ and rad51Δ .

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HU and MMS sensitivity of different mutant combinations of rpb1‐1, rpb1‐S751F, pol32Δ and rad51Δ.


Western blot against the phosphorylated form of histone H2A in the presence of 100 mM HU in asynchronously growing WT and RNAPII mutant cells . Ponceau staining is shown as a loading control .

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Western blot against the phosphorylated form of histone H2A in the presence of 100 mM HU in asynchronously growing WT and RNAPII mutant cells. Ponceau staining is shown as a loading control.


Rad52 foci formation at 30 and 37°C.


Recombination frequencies between inverted repeats in WT and RNAPII mutants measured with the plasmid ‐ born pTINV system . Recombinants were selected as Leu + .

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Recombination frequencies between inverted repeats in WT and RNAPII mutants measured with the plasmid‐born pTINV system. Recombinants were selected as Leu+.


Recombination frequencies using the direct ‐ repeat chromosomal system leu2 ‐ k : : ADE2 ‐ URA3 : : leu2 ‐ k in WT and RNAPII mutants . Deletion events were detected and quantified in media containing 5 ‐ fluorotic acid ( FOA ) and could be directly visualized as red sectors .

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Recombination frequencies using the direct‐repeat chromosomal system leu2‐k::ADE2‐URA3::leu2‐k in WT and RNAPII mutants. Deletion events were detected and quantified in media containing 5‐fluorotic acid (FOA) and could be directly visualized as red sectors.


Recombination frequencies between plasmid ‐ born direct repeats in WT and RNAPII mutants . The frequency of Leu + recombination was determined in the systems L ‐ lacZ and GL ‐ lacZ . Recombination frequencies are plotted as a function of the transcription levels . Low transcription refers to the GL ‐ lacZ systems in strains cultured in 2 % glucose ; medium refers to L ‐ lacZ in 2 % glucose , and high to GL ‐ lacZ in 2 % galactose .

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Recombination frequencies between plasmid‐born direct repeats in WT and RNAPII mutants. The frequency of Leu+ recombination was determined in the systems L‐lacZ and GL‐lacZ. Recombination frequencies are plotted as a function of the transcription levels. Low transcription refers to the GL‐lacZ systems in strains cultured in 2% glucose; medium refers to L‐lacZ in 2% glucose, and high to GL‐lacZ in 2% galactose.


Mitotic stability of centromeric plasmid pGAL ‐ lacZ in RNAPII mutants . Stability was analyzed in galactose after 23 generations . A small diagram of each system ( not drawn to scale ) is shown .

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Mitotic stability of centromeric plasmid pGAL‐lacZ in RNAPII mutants. Stability was analyzed in galactose after 23 generations. A small diagram of each system (not drawn to scale) is shown.


Recombination frequencies measured with the pTINV system in WT , rpb1 ‐ 1 , mre11Δ and rpb1 ‐ 1 mre11Δ .

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Recombination frequencies measured with the pTINV system in WT, rpb1‐1, mre11Δ and rpb1‐1 mre11Δ.


Rad52 focus formation in asynchronously growing cells at 30 ° C .

Rad52 focus formation in asynchronously growing cells at 30°C.


Viability after different times in 10 mM HU . The same cultures without HU were used as a control . The average values and standard deviations from three independent experiments are shown .

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Viability after different times in 10 mM HU. The same cultures without HU were used as a control. The average values and standard deviations from three independent experiments are shown.


Rad52 focus formation in asynchronously growing cells expressing normal or high levels of RNH1 .

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Rad52 focus formation in asynchronously growing cells expressing normal or high levels of RNH1.


FACS analysis of cell cycle progression in rpb mutants . Cells were synchronized in G1 with α ‐ factor and monitored at different times after release in the presence of 40 mM HU .

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FACS analysis of cell cycle progression in rpb mutants. Cells were synchronized in G1 with α‐factor and monitored at different times after release in the presence of 40 mM HU.