( C ) Left : Heat map showing the binding specificities ( Z - scores ) of selected Fab71 human analogous antibodies to human recPrP23 ‑ 230 compared to the negative controls ( BSA and Neu ) . Right : Heat map representing the reactivity ( - logEC50 ) obtained from dose - dependent ELISA binding curves of the analogous antibodies to human recPrP23 ‑ 230 . Red : low reactivity ; blue : high reactivity .

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

(C) Left: Heat map showing the binding specificities (Z-scores) of selected Fab71 human analogous antibodies to human recPrP23‑230 compared to the negative controls (BSA and Neu). Right: Heat map representing the reactivity (-logEC50) obtained from dose-dependent ELISA binding curves of the analogous antibodies to human recPrP23‑230. Red: low reactivity; blue: high reactivity.


( C ) Boxplot with half - violin plot displaying the distribution of autoantibody reactivity in 48 , 718 plasma samples . Twenty - seven samples over the reactivity threshold of - log ( EC50 ) ≥ 2 and fitting error < 20 % were considered as hits in the primary screen . Black dots correspond to single samples . The boxplot divides the data set into three quartiles . The box extends from the first to the third quartile values of the data set , with a line at the median . Whiskers show the range of the data ( minimum and maximum ) .

O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplot with half-violin plot displaying the distribution of autoantibody reactivity in 48,718 plasma samples. Twenty-seven samples over the reactivity threshold of -log(EC50) ≥ 2 and fitting error < 20% were considered as hits in the primary screen. Black dots correspond to single samples. The boxplot divides the data set into three quartiles. The box extends from the first to the third quartile values of the data set, with a line at the median. Whiskers show the range of the data (minimum and maximum).


( E ) Heat map representation for the validation of the hits from the primary screen ( 21 out of 27 hits ) . Confirmed hits displayed selective reactivity against human recPrP23 ‑ 230 . Controls : Patients # 11 - 13 , ApoE4 and BSA . Red : high reactivity ; blue : low reactivity .

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(E) Heat map representation for the validation of the hits from the primary screen (21 out of 27 hits). Confirmed hits displayed selective reactivity against human recPrP23‑230. Controls: Patients #11-13, ApoE4 and BSA. Red: high reactivity; blue: low reactivity.


( F ) Heat maps representing autoantibody clonality of hits analyzed by ELISA . Autoantibodies were mostly constituted by lambda light chains ( λ ) , while the kappa light chains ( κ ) showed prevalence in the total IgG fraction .

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(F) Heat maps representing autoantibody clonality of hits analyzed by ELISA. Autoantibodies were mostly constituted by lambda light chains (λ), while the kappa light chains (κ) showed prevalence in the total IgG fraction.


( G ) Determination of antibody reactivity ( - log ( EC50 ) ) for three individuals at two different time points . Individuals were selected by displaying strong anti - PrP IgG reactivity in the first test ( Time 1 ) . Reactivity was maintained for even more than one year ( Time 2 ) .

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(G) Determination of antibody reactivity (-log(EC50)) for three individuals at two different time points. Individuals were selected by displaying strong anti-PrP IgG reactivity in the first test (Time 1). Reactivity was maintained for even more than one year (Time 2).


( H ) Purified IgGs from patients with strong anti - PrP reactivity were tested against PrP variants and BSA ( negative control ) in dose - dependent ELISAs . Patient # 5 and # 6 showed binding mostly to the human recPrP121 ‑ 230 . Patient # 1 displayed a polyreactive pattern with binding to the human recPrP121 ‑ 230 and mouse recPrP23 - 110 .

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

(H) Purified IgGs from patients with strong anti-PrP reactivity were tested against PrP variants and BSA (negative control) in dose-dependent ELISAs. Patient #5 and #6 showed binding mostly to the human recPrP121‑230. Patient #1 displayed a polyreactive pattern with binding to the human recPrP121‑230 and mouse recPrP23-110.


RT - qPCR analysis of mRNA expression of indicated genes from MoLC or MoDC treated with TGF - β for 16h normalized to MoDC . Data represent mean values ± SD of triplicates from two donors .

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RT-qPCR analysis of mRNA expression of indicated genes from MoLC or MoDC treated with TGF-β for 16h normalized to MoDC. Data represent mean values ± SD of triplicates from two donors.


Subcellular localization analysis of S100A9 and S100A8 in MoLC , MoDC and MoDC - TGF - β . S100A9 mean fluorescence intensities ( MFI ) were normalized to MoLC and represented in the upper graph . The same was done for S100A9 cytoplasmic / nuclear MFI and shown in the lower graph . Scale bar : 5µm . Graphs show normalized percentages ± SD of data from four different donors for MoLC ( n = 4 ) and MoDC ( n = 4 ) and three different donors for TGF - β - treated MoDC ( n = 3 ) . When applicable , student t - test was performed to determine statistical significance * * P < 0 . 01 ) .

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

Subcellular localization analysis of S100A9 and S100A8 in MoLC, MoDC and MoDC-TGF-β. S100A9 mean fluorescence intensities (MFI) were normalized to MoLC and represented in the upper graph. The same was done for S100A9 cytoplasmic/nuclear MFI and shown in the lower graph. Scale bar: 5µm. Graphs show normalized percentages ± SD of data from four different donors for MoLC (n=4) and MoDC (n=4) and three different donors for TGF-β-treated MoDC (n=3). When applicable, student t-test was performed to determine statistical significance **P<0.01).


MoLC from six different donors ( n = 6 ) were left untreated ( NT ) or incubated with LPS for 48h and analysed for S100A9 expression by RT - qPCR ( left graph ) . MoLC treated as before were then transduced with HIV - GFP vectors for 48h and GFP + cells were analysed by flow cytometry and data were graphically represented as fold relative to non - treated MoLC ( right graph ) . Black horizontal line correspond to the mean . When applicable , student t - test was performed to determine statistical significance ( * * * P < 0 . 001 ) .

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

MoLC from six different donors (n=6) were left untreated (NT) or incubated with LPS for 48h and analysed for S100A9 expression by RT-qPCR (left graph). MoLC treated as before were then transduced with HIV-GFP vectors for 48h and GFP+ cells were analysed by flow cytometry and data were graphically represented as fold relative to non-treated MoLC (right graph). Black horizontal line correspond to the mean. When applicable, student t-test was performed to determine statistical significance (***P<0.001).


Cells transfected as above were pre - treated or not with nevirapine and transduced with HIV - GFP vectors for 48h . The percentage of GFP + MoLC was analysed by flow cytometry . Data represent means values ± SD from two different donors .

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Cells transfected as above were pre-treated or not with nevirapine and transduced with HIV-GFP vectors for 48h. The percentage of GFP+ MoLC was analysed by flow cytometry. Data represent means values ± SD from two different donors.


MoLC , dermal and epidermal cells were subjected to confocal immunofluorescence microscopy for langerin and S100A9 expression analysis . Scale bar : 5µm .

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MoLC, dermal and epidermal cells were subjected to confocal immunofluorescence microscopy for langerin and S100A9 expression analysis. Scale bar: 5µm.


Human epidermal cells treated as before were challenged for 48h with 20 and 200ng of p24 of HIV - Luc . When indicated cells were pre - treated with 10μM Nevirapine for 30 min before viral challenge . Luciferase activities measured in duplicates for each condition from two ( infection with 20 ng P24 ) or three ( infection with 200 ng P24 ) different skin samples were represented on a graph as means of relative luminescence units ( RLU ) ± SD .

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

Human epidermal cells treated as before were challenged for 48h with 20 and 200ng of p24 of HIV-Luc. When indicated cells were pre-treated with 10μM Nevirapine for 30 min before viral challenge. Luciferase activities measured in duplicates for each condition from two (infection with 20 ng P24) or three (infection with 200 ng P24) different skin samples were represented on a graph as means of relative luminescence units (RLU) ± SD.


The experiment shown is representative of four donors for HIV - 1 - R5 ( n = 4 ) and three donors for HIV - 1 primary isolates ( n = 3 ) . Data collected from all donors were compiled and represented as bar charts for cells challenged with HIV - 1 - R5 and HIV - 1 primary isolates . Nevirapine ( Nev ) ( 10μM ) was used as control when indicated and data were represented as means ± SD of HIV - Gag + cells from at least 2 ( for siS100A9 - MoLC infected by HIV - 1 primary isolates ) to 3 donors . When applicable , student t - test was performed to determine statistical significance ( * P < 0 . 1 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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The experiment shown is representative of four donors for HIV-1-R5 (n=4) and three donors for HIV-1 primary isolates (n=3). Data collected from all donors were compiled and represented as bar charts for cells challenged with HIV-1-R5 and HIV-1 primary isolates. Nevirapine (Nev) (10μM) was used as control when indicated and data were represented as means ± SD of HIV-Gag+ cells from at least 2 (for siS100A9-MoLC infected by HIV-1 primary isolates) to 3 donors. When applicable, student t-test was performed to determine statistical significance (*P<0.1;**P<0.01;***P<0.001).


E2C - and S100A9 - expressing HEK293T cells were infected with the indicated MOI of HIV - GFP for 48h and GFP + cells were analysed by flow cytometry . Mean values ± SD from four independent experiments ( n = 4 ) were normalized to the E2C - HEK293T control and represented in graph .

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E2C- and S100A9-expressing HEK293T cells were infected with the indicated MOI of HIV-GFP for 48h and GFP+ cells were analysed by flow cytometry. Mean values ± SD from four independent experiments (n=4) were normalized to the E2C-HEK293T control and represented in graph.


The percentage of HIV - Gag p24 + cells from four independent experiments for CEM ( n = 4 ) and two for Jurkat ( n = 2 ) analysed by flow cytometry were graphically represented as means of HIV - Gag + cells ± SD . When applicable , student t - test was performed to determine statistical significance ( * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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

The percentage of HIV-Gag p24+ cells from four independent experiments for CEM (n=4) and two for Jurkat (n=2) analysed by flow cytometry were graphically represented as means of HIV-Gag+ cells ± SD. When applicable, student t-test was performed to determine statistical significance (**P<0.01;***P<0.001).


E2C - and S100A9 - expressing CHO cells were challenged with indicated MOI of MMLV - YFP vectors for 24h or 48h . The percentages of YFP + CHO infected cells analysed by flow cytometry and data were represented on graphs .

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E2C- and S100A9-expressing CHO cells were challenged with indicated MOI of MMLV-YFP vectors for 24h or 48h. The percentages of YFP+ CHO infected cells analysed by flow cytometry and data were represented on graphs.


E2C - or S100A9 - expressing THP1 were left uninfected ( / ) or infected for 3 h with the indicated p24 amounts of HIV - 1 NL4 - 3 / Nef - IRES - Renilla viruses containing BlaM - VPR . Cells were processed for Blam - Vpr assay and the percentage of cleaved CCF2 was analysed by flow cytometry . The mock condition corresponds to unstained E2C - expressing cells . Dot - plots from a representative experiment are shown .

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

E2C- or S100A9-expressing THP1 were left uninfected (/) or infected for 3 h with the indicated p24 amounts of HIV-1 NL4-3/Nef-IRES-Renilla viruses containing BlaM-VPR. Cells were processed for Blam-Vpr assay and the percentage of cleaved CCF2 was analysed by flow cytometry. The mock condition corresponds to unstained E2C-expressing cells. Dot-plots from a representative experiment are shown.


Schematic description of fate - of - capsid assay is displayed above . HEK293T expressing E2C or S100A9 were transduced with HIV - Luc ( 10ng p24 ) for 4 h . Cells were then subjected to the fate of capsid assay ( see methods ) in order to pull - down intact cores ( CA cores ) ( recovered in the pellet ) . Equal volumes from the soluble ( top fraction ) and pelleted ( down fraction ) materials were analyzed by western blotting in order to evaluate the presence of p24 ( CA ) , CypA , S100A9 and E2C proteins .

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

Schematic description of fate-of-capsid assay is displayed above. HEK293T expressing E2C or S100A9 were transduced with HIV-Luc (10ng p24) for 4 h. Cells were then subjected to the fate of capsid assay (see methods) in order to pull-down intact cores (CA cores) (recovered in the pellet). Equal volumes from the soluble (top fraction) and pelleted (down fraction) materials were analyzed by western blotting in order to evaluate the presence of p24 (CA), CypA, S100A9 and E2C proteins.


Colorimetric HIV - 1 RTase activity was performed with 10µg of immunoprecipitated and eluted E2C or S100A9 in presence of increasing MgCl2 concentrations . Means ± SD colorimetric data obtained from at least three independent experiments were normalized to E2C - containing conditions and shown on graphs .

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Colorimetric HIV-1 RTase activity was performed with 10µg of immunoprecipitated and eluted E2C or S100A9 in presence of increasing MgCl2 concentrations. Means ± SD colorimetric data obtained from at least three independent experiments were normalized to E2C-containing conditions and shown on graphs.


TBH cytotoxicity assay in vivo of the ScAhp1 lysine mutants in yeast . Growth of yap1Δahp1Δ cells carrying empty vector ( ev ) , wild type peroxiredoxin gene ( AHP1 ) or indicated cysteine or lysine substitutions was monitored without or with 1 mM TBH . ScAhp14KR : ScAhp1C31S K47 / 48R K124R K156R ; ScAhp15KR : ScAhp1C31S K32R K47 / 48R K124R K156R .

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TBH cytotoxicity assay in vivo of the ScAhp1 lysine mutants in yeast. Growth of yap1Δahp1Δ cells carrying empty vector (ev), wild type peroxiredoxin gene (AHP1) or indicated cysteine or lysine substitutions was monitored without or with 1 mM TBH. ScAhp14KR: ScAhp1C31S K47/48R K124R K156R; ScAhp15KR: ScAhp1C31S K32R K47/48R K124R K156R.


In vivo conjugation analyses of ScAhp1 lysine mutants with protein extracts obtained from the indicated yeast strains expressing HA - URM1 . Unconjugated Urm1 and Urm1 - Ahp1 conjugates were detected by anti - HA ( top panels ) Western blots . Anti - Ahp1 blots ( middle ) detect unmodified Ahp1 . Anti - Cdc19 ( bottom panels ) served as loading control . ScAhp14KR : ScAhp1C31S K47 / 48R K124R K156R ; ScAhp15KR : ScAhp1C31S K32R K47 / 48R K124R K156R .

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In vivo conjugation analyses of ScAhp1 lysine mutants with protein extracts obtained from the indicated yeast strains expressing HA-URM1. Unconjugated Urm1 and Urm1-Ahp1 conjugates were detected by anti-HA (top panels) Western blots. Anti-Ahp1 blots (middle) detect unmodified Ahp1. Anti-Cdc19 (bottom panels) served as loading control. ScAhp14KR: ScAhp1C31S K47/48R K124R K156R; ScAhp15KR: ScAhp1C31S K32R K47/48R K124R K156R.


TBH cytotoxicity assay in vivo . Basic growth and the synthetic effect in wild type , ahp1Δ , urm1Δ , tum1Δ , ncs6Δ , cys3Δ , cys4Δ , cys3Δ urm1Δ , and cys4Δ urm1Δ strains was monitored without or with 1 mM TBH .

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TBH cytotoxicity assay in vivo. Basic growth and the synthetic effect in wild type, ahp1Δ, urm1Δ, tum1Δ, ncs6Δ, cys3Δ, cys4Δ, cys3Δ urm1Δ, and cys4Δ urm1Δ strains was monitored without or with 1 mM TBH.


H2S production using Biggy agar plates - Wild type , ahp1Δ , urm1Δ , tum1Δ , ncs6Δ , cys3Δ , cys4Δ , cys3Δ urm1Δ , and cys4Δ urm1Δ strains were grown on Biggy medium , and H2S production was

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H2S production using Biggy agar plates- Wild type, ahp1Δ, urm1Δ, tum1Δ, ncs6Δ, cys3Δ, cys4Δ, cys3Δ urm1Δ, and cys4Δ urm1Δ strains were grown on Biggy medium, and H2S production was recorded


In vivo Ahp1 conjugation analyses with protein extracts obtained from the indicated yeast strains expressing HA - URM1 . Unconjugated Urm1 and Urm1 - Ahp1 conjugates were detected by anti - HA ( top panels ) Western blots . Anti - Ahp1 blots ( middle ) detect unmodified Ahp1 . Anti - Cdc19 ( bottom panels ) served as loading control .

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

In vivo Ahp1 conjugation analyses with protein extracts obtained from the indicated yeast strains expressing HA-URM1. Unconjugated Urm1 and Urm1-Ahp1 conjugates were detected by anti-HA (top panels) Western blots. Anti-Ahp1 blots (middle) detect unmodified Ahp1. Anti-Cdc19 (bottom panels) served as loading control.


Northern - blot analysis for thiolation levels of tRNA < sub > UUC < / sub > < sup > Glu < / sup > in the genetic background of the indicated yeast strains . Total tRNA was resolved on denaturing PAGE supplemented with APM to retard the migration of thiolated tRNAUUCGlu . APM : ( [ N - Acryloyl - amino ] phenyl ) mercuric chloride . ( bottom ) Quantified thiolation levels from 3 independent biological replicates .

Northern-blot analysis for thiolation levels of tRNAUUCGlu in the genetic background of the indicated yeast strains. Total tRNA was resolved on denaturing PAGE supplemented with APM to retard the migration of thiolated tRNAUUCGlu. APM: ([N-Acryloyl-amino] phenyl) mercuric chloride. (bottom) Quantified thiolation levels from 3 independent biological replicates.


Ferrozine - based intracellular iron concentration measurement in L6 cells after temporal analysis of iron treatment at 100 or 250 µM for multiple time points . * P < 0 . 05 ( unpaired Student ' s t - test versus each time points with FeSO4 100 µM ) .

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Ferrozine-based intracellular iron concentration measurement in L6 cells after temporal analysis of iron treatment at 100 or 250 µM for multiple time points. *P < 0.05 (unpaired Student's t-test versus each time points with FeSO4 100 µM).


Representative confocal microscope images of L6 cells stained with IP - 1 ( Iron Probe 1 ) or transfected with IRE - CFP ( Iron Regulatory Element ) reporter after iron treatment ( FeSO4 , 250 µM ) for 24 h .

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

Representative confocal microscope images of L6 cells stained with IP-1 (Iron Probe 1) or transfected with IRE-CFP (Iron Regulatory Element) reporter after iron treatment (FeSO4, 250 µM) for 24 h.


Representative confocal images of L6 cells using iron sensitive fluorescent PGSK dye after iron treatment ( 250 µM , 24 h ) with iron chelator DPD ( 500 µM ) .

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

Representative confocal images of L6 cells using iron sensitive fluorescent PGSK dye after iron treatment (250 µM, 24 h) with iron chelator DPD (500 µM).


Relative gene expressions - ferritin heavy chain ( FTH ) , ferritin light chain ( FTL ) , ferroportin ( SLC40A1 , transferrin receptor 1 ( tfr1 , TFRC ) - normalized to 18sr RNA expression after iron treatment ( FeSO4 , 250 µM ) for 24 h . * P < 0 . 05 ( unpaired Student ' s t - test versus Basal ) .

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

Relative gene expressions - ferritin heavy chain (FTH), ferritin light chain (FTL), ferroportin (SLC40A1, transferrin receptor 1 (tfr1, TFRC) - normalized to 18sr RNA expression after iron treatment (FeSO4, 250 µM) for 24 h. *P < 0.05 (unpaired Student's t-test versus Basal).


Representative western blot images and quantification of ferritin ( heavy chain ) and tfr1 ( indicated with arrowhead ) over GAPDH after iron treatment ( 250 µM , 24 h ) .

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

Representative western blot images and quantification of ferritin (heavy chain) and tfr1 (indicated with arrowhead) over GAPDH after iron treatment (250 µM, 24 h).


Glucose uptake of L6 cells with insulin stimulation ( 10 nM or 100 nM , 20 min ) after iron treatment ( 250 µM , 1 h or 24 h ) .

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE 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

Glucose uptake of L6 cells with insulin stimulation (10 nM or 100 nM, 20 min) after iron treatment (250 µM, 1 h or 24 h).


Representative western blot images and quantification of phospho - IRS1 ( Y612 ) and phospho - AKT ( T308 ) over GAPDH with insulin stimulation ( 10 nM or 100 nM , 5 min ) after iron treatment ( 250 µM , 24 h ) .

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

Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) over GAPDH with insulin stimulation (10 nM or 100 nM, 5 min) after iron treatment (250 µM, 24 h).


Representative confocal microscope image of L6 cells stably expressing tandem fluorescent - eGFP - mCherry LC3B treated with iron ( FeSO4 , 250 µM ) for the indicated time points ( 4 h , 8 h , 24 h ) . Quantification of mean LC3B puncta per cell from ( A ) . Experiments were repeated three times , and one representative experiment is presented here .

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

Representative confocal microscope image of L6 cells stably expressing tandem fluorescent-eGFP-mCherry LC3B treated with iron (FeSO4, 250 µM) for the indicated time points (4 h, 8 h, 24 h). Quantification of mean LC3B puncta per cell from (A). Experiments were repeated three times, and one representative experiment is presented here.


Representative western blot images and quantification of p62 to actin in L6 cells after iron treatment ( FeSO4 , 250 µM ) at multiple time points .

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

Representative western blot images and quantification of p62 to actin in L6 cells after iron treatment (FeSO4, 250 µM) at multiple time points.


Representative western blot images and quantification of LC3B - II to GAPDH in L6 cells after 4h iron treatment stimulated with chloroquine ( CQ , 30 µM ) . * Representative western blot images and quantification of LC3B - II to GAPDH in L6 cells after 24 h iron treatment stimulated with CQ 30 µM .

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

Representative western blot images and quantification of LC3B-II to GAPDH in L6 cells after 4h iron treatment stimulated with chloroquine (CQ, 30 µM). * Representative western blot images and quantification of LC3B-II to GAPDH in L6 cells after 24 h iron treatment stimulated with CQ 30 µM.


Representative confocal microscope z - stack image of mCherry - LC3B L6 cells after iron treatment ( 250 µM , 24 h ) .

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

Representative confocal microscope z-stack image of mCherry-LC3B L6 cells after iron treatment (250 µM, 24 h).


Quantification of LC3B puncta size from ( F ) . Experiments were performed three times , and one representative experiment is presented here .

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Quantification of LC3B puncta size from (F). Experiments were performed three times, and one representative experiment is presented here.


Quantification of autophagosomes motility from live cell microscopy ( Supplementary Movie 1 ) from mCherry - LC3B L6 cells under basal , iron ( 250 µM , 24 h ) and starvation ( amino acid free ) condition . Experiments were performed three times , and one representative experiment is presented here . * P < 0 . 05 ( unpaired Student ' s t - test compared to Iron 24h ) .

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

Quantification of autophagosomes motility from live cell microscopy (Supplementary Movie 1) from mCherry-LC3B L6 cells under basal, iron (250 µM, 24 h) and starvation (amino acid free) condition. Experiments were performed three times, and one representative experiment is presented here. *P < 0.05 (unpaired Student's t-test compared to Iron 24h).


Representative TEM images of autophagosome and autolysosomes in L6 cells after iron treatment ( 250 µM , 24 h ) .

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

Representative TEM images of autophagosome and autolysosomes in L6 cells after iron treatment (250 µM, 24 h).


Representative confocal microscope images of eGFP - mCherry - LC3B L6 cells with Lysotracker DeepRed after iron treatment ( 250 µM , 24 h ) .

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

Representative confocal microscope images of eGFP-mCherry-LC3B L6 cells with Lysotracker DeepRed after iron treatment (250 µM, 24 h).


Representative western blot images of phospho - ULK1 ( S757 ) , phospho - S6Kp70 ( T389 ) , total S6K , GAPDH after iron treatment ( FeSO4 , 250 µM ) for multiple time points . Quantification of mTORC1 activity analyzed through phosphorylation of S6K T389 to total S6k and ULK1 S757 to GAPDH after iron treatment ( FeSO4 , 250 µM ) for multiple time points .

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

Representative western blot images of phospho-ULK1 (S757), phospho-S6Kp70 (T389), total S6K, GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points. Quantification of mTORC1 activity analyzed through phosphorylation of S6K T389 to total S6k and ULK1 S757 to GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points.


Representative epi - immunofluorescent images of L6 cells transfected with myc - RHEB Q46L and immuno - stained against LC3B and myc after iron treatment ( FeSO4 , 250 µM ) for 24 h .

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

Representative epi-immunofluorescent images of L6 cells transfected with myc-RHEB Q46L and immuno-stained against LC3B and myc after iron treatment (FeSO4, 250 µM) for 24 h.


Ferrozine - based colorimetric measurement of intracellular iron in wild - type ( wt ) L6 and RHEB - Q64L L6 cells after iron treatment ( 50 µM or 250 µM ) for 24 h .

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Ferrozine-based colorimetric measurement of intracellular iron in wild-type (wt) L6 and RHEB-Q64L L6 cells after iron treatment (50 µM or 250 µM) for 24 h.


Representative confocal images of wt L6 and RHEB - Q64L L6 cells pulsed with Lysotracker Deep Red and immuno - stained against LC3B after iron treatment ( 250 µM , 24 h ) .

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

Representative confocal images of wt L6 and RHEB-Q64L L6 cells pulsed with Lysotracker Deep Red and immuno-stained against LC3B after iron treatment (250 µM, 24 h).


Quantification of LC3B - free lysosome numbers in ( E ) . Experiments were repeated three times , and one representative experiment is presented here . * P < 0 . 05 ( multiple unpaired Student ' s t - test versus Basal in wt and RHEB Q64L cells )

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

Quantification of LC3B-free lysosome numbers in (E). Experiments were repeated three times, and one representative experiment is presented here. *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells)


Representative TEM images of wt L6 and RHEB - Q64L cells after iron treatment ( 250 µM , 24 h ) .

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

Representative TEM images of wt L6 and RHEB-Q64L cells after iron treatment (250 µM, 24 h).


Representative western blot images and quantifications of phospho - IRS1 ( Y612 , indicated by arrowhead ) and AKT ( T308 , indicated by arrowhead ) to GAPDH in wt and RHEB - Q64L L6 cells stimulated with insulin ( 100 nM , 5 min ) after iron treatment ( 250 µM , 24 h ) . * P < 0 . 05 ( multiple unpaired Student ' s t - test versus Basal in wt and RHEB Q64L cells )

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

Representative western blot images and quantifications of phospho-IRS1 (Y612, indicated by arrowhead) and AKT (T308, indicated by arrowhead) to GAPDH in wt and RHEB-Q64L L6 cells stimulated with insulin (100 nM, 5 min) after iron treatment (250 µM, 24 h). *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells)


Representative confocal microscope images of L6 cells pulsed with PGSK dye after iron treatment ( 250 µM , 24 h ) followed by 24 h iron withdrawal . Ferrozine based colorimetric measurement of intracellular iron in L6 cells after iron treatment ( 250 µM , 24 h ) followed by 24 h withdrawal .

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

Representative confocal microscope images of L6 cells pulsed with PGSK dye after iron treatment (250 µM, 24 h) followed by 24 h iron withdrawal. Ferrozine based colorimetric measurement of intracellular iron in L6 cells after iron treatment (250 µM, 24 h) followed by 24 h withdrawal.


Representative western blot images and quantification of phospho - IRS1 ( Y612 ) and phospho - AKT ( T308 ) to GAPDH in L6 cells iron treatment ( 250 µM , 24 h ) followed by 24 h withdrawal .

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

Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in L6 cells iron treatment (250 µM, 24 h) followed by 24 h withdrawal.


Representative epi - immunofluorescent microscope images of L6 cells immuno - stained against LC3B and LAMP1 after iron treatment ( 250 µM , 24 h ) followed by 3 h withdrawal .

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

Representative epi-immunofluorescent microscope images of L6 cells immuno-stained against LC3B and LAMP1 after iron treatment (250 µM, 24 h) followed by 3 h withdrawal.


Quantification of autophagosome - free lysosomes in ( E ) . Experiments were performed three times , and one representative experiment is presented here .

O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O

Quantification of autophagosome-free lysosomes in (E). Experiments were performed three times, and one representative experiment is presented here.


Representative western blot images and quantification of ferritin to vinculin in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h or 24 h .

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

Representative western blot images and quantification of ferritin to vinculin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h or 24 h.


Representative western blot images and quantification of phospho - mTOR S2448 , total mTOR and actin in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h .

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

Representative western blot images and quantification of phospho-mTOR S2448, total mTOR and actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h.


Representative western blot images of phospho - mTOR S2448 , total mTOR phsophpo - TSC2 T1462 , total TSC2 , phospho - AKT S473 , total AKT , actin in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without MK2206 ( AKT inhibitor . )

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

Representative western blot images of phospho-mTOR S2448, total mTOR phsophpo-TSC2 T1462, total TSC2, phospho-AKT S473, total AKT, actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without MK2206 (AKT inhibitor.)


Representative western blot images of phospho - UVRAG S550 and total UVRAG in L6 cells transfected with FLAG - UVRAG and FLAG pulldown after iron treatment ( 250 µM , 4 h and 24 h ) followed by withdrawal for 1 h and 3 h .

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

Representative western blot images of phospho-UVRAG S550 and total UVRAG in L6 cells transfected with FLAG-UVRAG and FLAG pulldown after iron treatment (250 µM, 4 h and 24 h) followed by withdrawal for 1 h and 3 h.


Representative western blot images of endogenous phospho - UVRAG S550 , total UVRAG expression in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without torin1 ( 200 nM ) .

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

Representative western blot images of endogenous phospho-UVRAG S550, total UVRAG expression in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without torin1 (200 nM).


Live cell imaging analysis of L6 cells transfected with LAMP1 - RFP from Movie EV2 : lysosomal number in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without Torin1 ( 200 nM ) .

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

Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: lysosomal number in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM).


Live cell imaging analysis of L6 cells transfected with LAMP1 - RFP from Movie EV2 : speed ( F ) in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without Torin1 ( 200 nM ) .

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

Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: speed (F) in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM).


Representative confocal images of time - lapse captures of lysosomes at 6 sec intervals for 30 sec . White and yellow arrows mark positions of two representative LAMP1 puncta over 30 seconds , and the traces of puncta were shown as lines in final panel .

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

Representative confocal images of time-lapse captures of lysosomes at 6 sec intervals for 30 sec. White and yellow arrows mark positions of two representative LAMP1 puncta over 30 seconds, and the traces of puncta were shown as lines in final panel.


Prussian Perl blue staining in liver and skeletal muscle after iron injections .

Prussian Perl blue staining in liver and skeletal muscle after iron injections.


Representative western blot images and quantification of ferritin and tfr1 ( indicated by arrowhead ) to tubulin in skeletal muscles 24 h after iron injections .

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

Representative western blot images and quantification of ferritin and tfr1 (indicated by arrowhead) to tubulin in skeletal muscles 24 h after iron injections.


Insulin tolerance test 24 h after iron injections .

Insulin tolerance test 24 h after iron injections.


Glucose tolerance test ( GTT ) 24 h after iron injections .

Glucose tolerance test (GTT) 24 h after iron injections.


Insulin tolerance test 24 h after iron injections . Quantification of area under curve in ITT ( E ) .

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Insulin tolerance test 24 h after iron injections. Quantification of area under curve in ITT (E).


Glucose tolerance test ( GTT ) 24 h after iron injections . Quantification of area under curve for panel ( F ) .

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

Glucose tolerance test (GTT) 24 h after iron injections. Quantification of area under curve for panel (F).


Representative western blot images and quantification of phospho - IRS1 ( Y612 ) and phospho - AKT ( T308 ) to GAPDH in skeletal muscles 24 h after iron injection followed by i . p . insulin injection .

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

Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in skeletal muscles 24 h after iron injection followed by i.p. insulin injection.


Representative confocal microscope images of skeletal muscle tissue sections immuno - stained against LC3B ( Alexa 647 ) and LAMP1 ( Alexa 555 ) Quantification of lysosomes ( LAMP1 puncta ) in skeletal muscles 24 h after iron injections .

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

Representative confocal microscope images of skeletal muscle tissue sections immuno-stained against LC3B (Alexa 647) and LAMP1 (Alexa 555) Quantification of lysosomes (LAMP1 puncta) in skeletal muscles 24 h after iron injections.


Representative TEM images of skeletal muscle 24 h after iron injections .

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Representative TEM images of skeletal muscle 24 h after iron injections.


Representative western blot images of phospho - UVRAG S550 and total UVRAG in skeletal muscle 24 h after iron injections .

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

Representative western blot images of phospho-UVRAG S550 and total UVRAG in skeletal muscle 24 h after iron injections.


Representative western blot images and quantification of LC3 - B and p62 to tubulin in skeletal muscles 24 h after iron injections .

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Representative western blot images and quantification of LC3-B and p62 to tubulin in skeletal muscles 24 h after iron injections.


( A ) Example regions exhibiting the IPOD - HR occupancy where EPOD calls are made . Loose EPODs ( pink ) and Strict EPODs ( teal ) represent spans of high protein occupancy at lower and higher thresholds , respectively . Neighboring areas showing lower robust z scores are not EPODs . Note that following the definitions in [ 12 ] , the presence of an EPOD requires a sustained region of high average occupancy , without any regions in which occupancy drops to background levels . Thus , breaks in EPOD calls represent cases where a rolling average occupancy drops below the calling threshold ( e . g . , the breakpoint in the middle of pinQ in the top panel ) , and regions of apparent occupancy may nevertheless not be called due to being below the minimum length threshold ( as is the case for nohA ) .

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

(A) Example regions exhibiting the IPOD-HR occupancy where EPOD calls are made. Loose EPODs (pink) and Strict EPODs (teal) represent spans of high protein occupancy at lower and higher thresholds, respectively. Neighboring areas showing lower robust z scores are not EPODs. Note that following the definitions in [12], the presence of an EPOD requires a sustained region of high average occupancy, without any regions in which occupancy drops to background levels. Thus, breaks in EPOD calls represent cases where a rolling average occupancy drops below the calling threshold (e.g., the breakpoint in the middle of pinQ in the top panel), and regions of apparent occupancy may nevertheless not be called due to being below the minimum length threshold (as is the case for nohA).


( E ) Specific locations across all conditions remain occupied by protein where EPODs across a representative region , the waa operon , were called ( left ) and unoccupied by RNA polymerase ( right ) . The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD - HR occupancy .

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(E) Specific locations across all conditions remain occupied by protein where EPODs across a representative region, the waa operon, were called (left) and unoccupied by RNA polymerase (right). The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy.


( A ) Number of EPODs called in different NAP deletions . D . S . denotes genotypes where cells were collected in the deep stationary phase of growth .

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(A) Number of EPODs called in different NAP deletions. D.S. denotes genotypes where cells were collected in the deep stationary phase of growth.


( D ) Distributions of mean protein occupancy at WT EPOD boundaries and background . The blue dots denote the median and the black line displays the interquartile ranges in each condition . The dashed pink line represents the WT median . ( * ) indicate the Wilcoxon Rank Sum test p value comparing the change in pseudomedian vs . WT for each condition that has been adjusted using the Benjamini and Hochberg method ( against a null hypothesis of no difference in pseudomedians ) . The grey line denotes the same comparison between the D . S . conditions . Underlined ( * ) ' s indicate a gain in the pseudomedian compared to baseline conditions , while no underline indicates a loss in the pseudomedian compared to baseline conditions . p value < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * * . Three biological replicates were used for the " WT , Rich " case and " WT ( deep stat . ) " case , and two biological replicates for the " WT , Min " and " WT ( 2 ) , Rich " conditions .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Distributions of mean protein occupancy at WT EPOD boundaries and background. The blue dots denote the median and the black line displays the interquartile ranges in each condition. The dashed pink line represents the WT median. (*) indicate the Wilcoxon Rank Sum test p value comparing the change in pseudomedian vs. WT for each condition that has been adjusted using the Benjamini and Hochberg method (against a null hypothesis of no difference in pseudomedians). The grey line denotes the same comparison between the D.S. conditions. Underlined (*)'s indicate a gain in the pseudomedian compared to baseline conditions, while no underline indicates a loss in the pseudomedian compared to baseline conditions. p value < 0.05 = *, <0.005=**, <0.0005=***. Three biological replicates were used for the "WT, Rich" case and "WT (deep stat.)" case, and two biological replicates for the "WT, Min" and "WT (2), Rich" conditions.


( E ) Protein occupancy over the waa operon . The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD - HR occupancy . The EPOD over the waa operon is lost in the ∆ stpA ∆ hns condition which results in increased accessibility and RNA polymerase

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(E) Protein occupancy over the waa operon. The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. The EPOD over the waa operon is lost in the ∆stpA∆hns condition which results in increased accessibility and RNA polymerase occupancy


( G ) RNA - seq analysis shows log fold change compared to WT of waa operon expression upon deletion of hns and stpA . ( * ) indicate q value < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * *

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(G) RNA-seq analysis shows log fold change compared to WT of waa operon expression upon deletion of hns and stpA. (*) indicate q value < 0.05 = *, < 0.005 = **, < 0.0005 = ***


( B ) Protein occupancy over the idn operon for each condition ( colors are denoted in ( A ) ) . The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD - HR occupancy . There is a large loss in protein occupancy when cells are shifted to 5KDG , leading to the loss of the call ed EPOD . Protein occupancy is restored once cells are grown in the second glucose condition . The 500bp normalized average of previously published H - NS ChIP - seq [ 20 ] exhibits high H - NS binding on the idnD promoter region .

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(B) Protein occupancy over the idn operon for each condition (colors are denoted in (A)). The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. There is a large loss in protein occupancy when cells are shifted to 5KDG, leading to the loss of the call ed EPOD. Protein occupancy is restored once cells are grown in the second glucose condition. The 500bp normalized average of previously published H-NS ChIP-seq [20] exhibits high H-NS binding on the idnD promoter region.


( C ) To examine the expression of the idn operon at each shift , RNA - seq was performed . RNA - seq expression estimates log2 scaled for idn operon genes for WT cells grown in each condition ( as colored in ( A ) ) . Comparisons are denoted with colored dots with significance stars representing the adjusted p - value where ( * * * ) signify q - values < 0 .

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

(C) To examine the expression of the idn operon at each shift, RNA-seq was performed. RNA-seq expression estimates log2 scaled for idn operon genes for WT cells grown in each condition (as colored in (A)). Comparisons are denoted with colored dots with significance stars representing the adjusted p-value where (***) signify q-values <0.0005


( B ) Difference in Log2 ratio of fitnesses of the KDG - exposed cells with unexposed lacZ : : cat relative to that observed prior to KDG exposure , given as a function of time since exposure to KDG . Day - 1 corresponds to the day before KDG exposure , Day 0 is cells taken immediately after growth in KDG , and subsequent timepoints reflect different durations of growth in M9 Min Glu prior to competition ( See ( A ) ) . Points show medians across replicates ( 3 biological replicates for 0 . 5 days , 6 biological replicates for all other timepoints ) ; error bars show 95 % confidence intervals for the difference relative to the Day - 1 timepoint , calculated using the R wilcox . test function . Significance was assessed using a Wilcoxon rank sum test comparing the distribution at each timepoint to the - 1 day ( naive ) timepoint : * , p < 0 . 05 ; * * , p < 0 . 005 .

O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Difference in Log2 ratio of fitnesses of the KDG-exposed cells with unexposed lacZ::cat relative to that observed prior to KDG exposure, given as a function of time since exposure to KDG. Day -1 corresponds to the day before KDG exposure, Day 0 is cells taken immediately after growth in KDG, and subsequent timepoints reflect different durations of growth in M9 Min Glu prior to competition (See (A)). Points show medians across replicates (3 biological replicates for 0.5 days, 6 biological replicates for all other timepoints); error bars show 95% confidence intervals for the difference relative to the Day -1 timepoint, calculated using the R wilcox.test function. Significance was assessed using a Wilcoxon rank sum test comparing the distribution at each timepoint to the -1 day (naive) timepoint: *, p<0.05; **, p<0.005.


( C ) Mean protein occupancy was calculated across all WT EPOD locations that contain annotated prophages . The change in mean protein occupancy compared to WT was calculated for each condition , where anything negative is a loss in occupancy compared to WT . Hierarchical clustering was performed to examine which genotypes clustered together and were more similar . Permutation tests comparing the change in occupancy over EPODs containing prophages were performed against the rest of the genome . P value < 0 . 05 = * , < 0 . 005 = * * indicate a negative mean change in occupancy compared to WT .

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(C) Mean protein occupancy was calculated across all WT EPOD locations that contain annotated prophages. The change in mean protein occupancy compared to WT was calculated for each condition, where anything negative is a loss in occupancy compared to WT. Hierarchical clustering was performed to examine which genotypes clustered together and were more similar. Permutation tests comparing the change in occupancy over EPODs containing prophages were performed against the rest of the genome. P value <0.05 = *, <0.005=** indicate a negative mean change in occupancy compared to WT.


( A ) Example prophage region that is annotated with the Fis - and Hfq - associated HMM class 5 in our genome - wide HMM classification The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD - HR occupancy . Prophage genes are highlighted with a red box . The major peak associated with WT IPOD - HR occupancy is represented in a grey dashed line as a reference to compare IPOD - HR occupancy in the other genotypes . Modest loss of protein occupancy was observed at the same prophage - containing EPOD for ∆ fis ( light purple ; see color key in ( B ) ) and ∆ hfq ( dark purple ; see color key in ( B ) ) conditions compared to WT ( gold ) .

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

(A) Example prophage region that is annotated with the Fis- and Hfq-associated HMM class 5 in our genome-wide HMM classification The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. Prophage genes are highlighted with a red box. The major peak associated with WT IPOD-HR occupancy is represented in a grey dashed line as a reference to compare IPOD-HR occupancy in the other genotypes. Modest loss of protein occupancy was observed at the same prophage-containing EPOD for ∆fis (light purple; see color key in (B)) and ∆hfq (dark purple; see color key in (B)) conditions compared to WT (gold).


( B ) RNA - seq of WT , ∆ fis , and ∆ hfq were performed . The log fold change compared to WT was calculated at prophage genes contained in the dashed box in ( A ) . Induction of prophages across the region where loss in occupancy is observed . ( * ) indicate the adjusted p - value : < 0 . 10 = + , < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * *

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD 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 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) RNA-seq of WT, ∆fis, and ∆hfq were performed. The log fold change compared to WT was calculated at prophage genes contained in the dashed box in (A). Induction of prophages across the region where loss in occupancy is observed. (*) indicate the adjusted p-value: <0.10 = +, < 0.05 = *, <0.005=**, <0.0005=***


( F ) Rate ratios of all genes ( grey ) and prophage genes ( red ) in each quadrant outline in ( E ) , showing a higher rate of genes that resided in the + / + category , indicating that many prophages are de - repressed in both ∆ fis and ∆ hfq . ( * ) indicate the p - value calculated from testing the null that the rate ratios are the same . P - value < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * *

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(F) Rate ratios of all genes (grey) and prophage genes (red) in each quadrant outline in (E), showing a higher rate of genes that resided in the +/+ category, indicating that many prophages are de-repressed in both ∆fis and ∆hfq. (*) indicate the p-value calculated from testing the null that the rate ratios are the same. P-value < 0.05 = *, <0.005=**, <0.0005=***


( G ) P1 vir transduction experiment to test the viability of ∆ fis and ∆ hfq . - Hfq indicates deleting Hfq and - CspE indicates deleting - CspE as a control . Strain identities are indicated in the box . Number of transductions were counted on LB + Kan plates ; all efficiencies are relative to WT , and thus the log - scaled relative transduction rate for the WT itself is 0 by definition . - R1 indicates that the prophage region in ( A ) was deleted to test whether the loss of prophages silenced by Fis and Hfq restored viability of a ∆ fis ∆ hfq genotype . R2 and R3 were other regions in the genome that contained prophages that appeared to have Fis / Hfq dependent EPODs . Plotted values are mean efficiencies across replicates , with error bars showing a 95 % credible interval obtained via Bayesian inference , assuming the replicate - level colony counts are Poisson distributed with a ( conjugate ) Gamma ( 0 , 0 ) prior ; all log ratios ( including error bounds ) are plotted relative to the mean WT value . Data obtained from 5 biological replicates for hfq : : kan transductions and 4 biological replicates for cspE : : kan transductions .

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

(G) P1 vir transduction experiment to test the viability of ∆fis and ∆hfq. -Hfq indicates deleting Hfq and -CspE indicates deleting -CspE as a control. Strain identities are indicated in the box. Number of transductions were counted on LB + Kan plates; all efficiencies are relative to WT, and thus the log-scaled relative transduction rate for the WT itself is 0 by definition. -R1 indicates that the prophage region in (A) was deleted to test whether the loss of prophages silenced by Fis and Hfq restored viability of a ∆fis ∆hfq genotype. R2 and R3 were other regions in the genome that contained prophages that appeared to have Fis/Hfq dependent EPODs. Plotted values are mean efficiencies across replicates, with error bars showing a 95% credible interval obtained via Bayesian inference, assuming the replicate-level colony counts are Poisson distributed with a (conjugate) Gamma(0,0) prior; all log ratios (including error bounds) are plotted relative to the mean WT value. Data obtained from 5 biological replicates for hfq::kan transductions and 4 biological replicates for cspE::kan transductions.


( A ) IPOD - HR ChIP - subtracted z - scores and RNA polymerase ChIP in the vicinity of an extended protein occupancy domain ( EPOD ) . ChIP tracks are shown as log2 extracted / input ratios ; the z - score is the ChIP - subtracted robust z - score smoothed with a 512 bp rolling median . ( B ) IPOD - HR and RNA polymerase ChIP tracks in the vicinity of a negative EPOD ( nEPOD ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O

(A) IPOD-HR ChIP-subtracted z-scores and RNA polymerase ChIP in the vicinity of an extended protein occupancy domain (EPOD). ChIP tracks are shown as log2 extracted/input ratios; the z-score is the ChIP-subtracted robust z-score smoothed with a 512 bp rolling median. (B) IPOD-HR and RNA polymerase ChIP tracks in the vicinity of a negative EPOD (nEPOD).


( C ) Effects of deletion of rok in the vicinity of an nEPOD ; Rok ChIP data from [ 70 ] shows a strong overlap with the nEPOD boundary , whereas that occupancy region is lost in Δrok cells .

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(C) Effects of deletion of rok in the vicinity of an nEPOD; Rok ChIP data from[70] shows a strong overlap with the nEPOD boundary, whereas that occupancy region is lost in Δrok cells.


( E ) Comparison of IPOD occupancy and SMC ChIP occupancy in the vicinity of several typical EPODs .

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(E) Comparison of IPOD occupancy and SMC ChIP occupancy in the vicinity of several typical EPODs.


A . Optical coherence tomography ( OCT ) horizontal B - scan and thickness map of neovascular age related macular degeneration ( NV AMD ) patient with active lesions ( 1 ) , cystoid macular edema ( 2 ) , subretinal fibrin deposits ( 3 ) and serous retinal detachment ( 4 ) . B . Optical coherence tomography ( OCT ) horizontal B - scan and thickness map of control patient with a medium sized , stage 3 , full thickness macular hole ( 5 ) .

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A. Optical coherence tomography (OCT) horizontal B-scan and thickness map of neovascular age related macular degeneration (NV AMD) patient with active lesions (1), cystoid macular edema (2), subretinal fibrin deposits (3) and serous retinal detachment (4). B. Optical coherence tomography (OCT) horizontal B-scan and thickness map of control patient with a medium sized, stage 3, full thickness macular hole (5).


C - G . Vitreous humor analyzed by ELISA for VEGF - A ( C ) ; n = 7 ( Ctrl ) , 9 ( NV AMD ) , SEMA3A ( D ) ; n = 10 ( Ctrl ) , 10 ( NV AMD ) , TGFβ ( E ) ; n = 8 ( Ctrl ) , 7 ( NV AMD ) , PDGF - BB ( F ) ; n = 5 ( Ctrl ) , 6 ( NV AMD ) , PGF ( G ) : n = 5 ( Ctrl ) , 5 ( NV AMD ) . Dots represent concentrations of individual patient samples .

O O O O B-TISSUE I-TISSUE O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-DISEASE I-DISEASE O O B-GENEPROD O O O O O O O O O O O O O B-DISEASE I-DISEASE O O B-GENEPROD O O O O O O O O O O O O O B-DISEASE I-DISEASE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-DISEASE I-DISEASE O O B-GENEPROD O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O B-ORGANISM O O

C-G. Vitreous humor analyzed by ELISA for VEGF-A (C); n = 7 (Ctrl), 9 (NV AMD), SEMA3A (D); n = 10 (Ctrl), 10 (NV AMD), TGFβ (E); n = 8 (Ctrl), 7 (NV AMD), PDGF-BB (F); n = 5 (Ctrl),6 (NV AMD), PGF (G): n = 5 (Ctrl), 5 (NV AMD). Dots represent concentrations of individual patient samples.


H . Representative confocal images of IBA - 1 - stained mononuclear phagocytes on choroidal flat mounts from LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl mice at D7 and D14 . Examples of macrophage quantification ( yellow stars ) are presented in side panels . Scale bar : 20μm . I , J . Total number of IBA - 1 - positive mononuclear phagocytes counted around laser impact area on confocal images of choroidal flat mounts at D7 ( I ) and D14 ( J ) ; n = 19 burns ( D7 LysM - Cre / Nrp1 + / + ) , n = 25 burns ( D7 LysM - Cre / Nrp1fl / fl ) n = 37 burns ( D7 LysM - Cre / Nrp1 + / + ) , n = 23 burns ( D7 LysM - Cre / Nrp1fl / fl ) , 3 - 5 mice with ~ 4 burns per eye .

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

H. Representative confocal images of IBA-1-stained mononuclear phagocytes on choroidal flat mounts from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D7 and D14. Examples of macrophage quantification (yellow stars) are presented in side panels. Scale bar: 20μm. I, J. Total number of IBA-1-positive mononuclear phagocytes counted around laser impact area on confocal images of choroidal flat mounts at D7 (I) and D14 (J); n = 19 burns (D7 LysM-Cre/Nrp1+/+), n = 25 burns (D7 LysM-Cre/Nrp1fl/fl) n = 37 burns (D7 LysM-Cre/Nrp1+/+), n = 23 burns (D7 LysM-Cre/Nrp1fl/fl), 3-5 mice with ~4 burns per eye.


K . Compilation of representative compressed Z - stack confocal images of FITC - dextran - labeled CNV and IB4 - stained laser impact area from LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl mice at D14 . Scale bar : 20μm . L - N . Quantification of area of FITC - dextran - labeled CNV ( L ) , isolectin B4 ( IB4 ) - stained laser impact area ( M ) and the ratio of FITC / IB4 per laser - burn ( N ) relative to LysM - Cre / Nrp1 + / + at D14 ; n = 23 burns ( LysM - Cre / Nrp1 + / + ) , n = 27 burns ( LysM - Cre / Nrp1fl / fl ) .

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

K. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and IB4-stained laser impact area from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14. Scale bar: 20μm. L-N. Quantification of area of FITC-dextran-labeled CNV (L), isolectin B4 (IB4 )-stained laser impact area (M) and the ratio of FITC/IB4 per laser-burn (N) relative to LysM-Cre/Nrp1+/+ at D14; n = 23 burns (LysM-Cre/Nrp1+/+), n = 27 burns ( LysM-Cre/Nrp1fl/fl).


A - D . mRNA expression of inflammation markers relative to LysM - Cre / Nrp1 + / + in mouse RPE - choroid - sclera complexes at D3 for Il1b ( A ) ; n = 10 ( LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl ) , Il6 ( B ) ; n = 7 ( LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl ) , Vegfa ( C ) ; n = 10 ( LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl ) , Tnf ( D ) ; n = 5 ( LysM - Cre / Nrp1 + / + ) , n = 10 ( LysM - Cre / Nrp1fl / fl ) .

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

A-D. mRNA expression of inflammation markers relative to LysM-Cre/Nrp1+/+ in mouse RPE-choroid-sclera complexes at D3 for Il1b (A); n = 10 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Il6 (B); n = 7 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Vegfa (C); n = 10 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Tnf (D); n = 5 (LysM-Cre/Nrp1+/+), n = 10 (LysM-Cre/Nrp1fl/fl).


K , L . mRNA expression relative to LysM - Cre / Nrp1 + / + of inflammation markers in mouse BMDMs for Il1b ( K ) ; n = 4 ( LysM - Cre / Nrp1 + / + ) , n = 5 ( LysM - Cre / Nrp1fl / fl ) and Il6 ( L ) ; n = 3 ( LysM - Cre / Nrp1 + / + ) , n = 2 ( LysM - Cre / Nrp1fl / fl )

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

K, L. mRNA expression relative to LysM-Cre/Nrp1+/+ of inflammation markers in mouse BMDMs for Il1b (K); n = 4 (LysM-Cre/Nrp1+/+), n = 5 (LysM-Cre/Nrp1fl/fl) and Il6 (L); n = 3 (LysM-Cre/Nrp1+/+), n = 2 (LysM-Cre/Nrp1fl/fl)


P . Heatmap ( left ) and enrichment plot ( right ) of GO Angiogenesis gene set enrichment analysis ( GSEA ) of wildtype and LysM - Cre / Nrp1fl / fl peritoneal macrophages ; n = 2 . NES , normalized enrichment score ; FDR , false discovery rate .

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

P. Heatmap (left) and enrichment plot (right) of GO Angiogenesis gene set enrichment analysis (GSEA) of wildtype and LysM-Cre/Nrp1fl/fl peritoneal macrophages; n = 2. NES, normalized enrichment score; FDR, false discovery rate.


E . Compilation of representative compressed Z - stack confocal images of FITC - dextran - labeled CNV and isolectin B4 ( IB4 ) - stained laser impact area from Vehicle and NRP1 - derived trap treated wildtype mice . Scale bar : 20μm . F - H . Quantification of area of FITC - dextran - labeled CNV ( F ) , IB4 - stained laser impact area ( G ) and the ratio of FITC / IB4 per laser - burn ( H ) relative to Vehicle at D14 ; n = 24 burns ( Vehicle ) , n = 26 burns ( NRP1 - derived trap ) .

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

E. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and isolectin B4 (IB4)-stained laser impact area from Vehicle and NRP1-derived trap treated wildtype mice. Scale bar: 20μm. F-H. Quantification of area of FITC -dextran-labeled CNV (F), IB4-stained laser impact area (G) and the ratio of FITC/IB4 per laser-burn (H) relative to Vehicle at D14; n = 24 burns (Vehicle), n = 26 burns (NRP1-derived trap).


J - L . Quantification of area of FITC - dextran - labeled CNV ( J ) , isolectin B4 ( IB4 ) - stained laser impact area ( K ) and the ratio of FITC / IB4 per laser - burn ( L ) relative to LysM - Cre / Nrp1 + / + + Vehicle in Vehicle and NRP1 - derived trap treated LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl mice at D14 ; n = 16 burns ( LysM - Cre / Nrp1 + / + + Vehicle ) , n = 16 burns ( LysM - Cre / Nrp1 + / + + Trap ) , n = 13 burns ( LysM - null / Nrp1fl / fl + Vehicle ) , n = 19 burns ( LysM - Cre / Nrp1fl / fl + Trap ) .

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

J-L. Quantification of area of FITC-dextran-labeled CNV (J), isolectin B4 (IB4)-stained laser impact area (K) and the ratio of FITC/IB4 per laser-burn (L) relative to LysM-Cre/Nrp1+/+ + Vehicle in Vehicle and NRP1-derived trap treated LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14; n = 16 burns (LysM-Cre/Nrp1+/+ + Vehicle), n = 16 burns (LysM-Cre/Nrp1+/+ + Trap), n = 13 burns (LysM-null/Nrp1fl/fl + Vehicle) , n = 19 burns (LysM-Cre/Nrp1fl/fl + Trap).


Representative PET , MRI and fused PET / MRI images of the thoracic region of a mouse that received the PDE10A radioligand [ 18F ] - AQ28A . Interscapular brown adipose tissue ( BAT ) in each image is highlighted by the crosshairs . The mean standardized uptake ( SUV ) value of [ 18F ] - AQ28A in BAT and skeletal muscle throughout the dynamic scan was calculated and compared for each animal ( n = 5 ) . * P = 0 . 0488 ( 20 mins ) , * P = 0 . 0236 ( 25 mins ) , * P = 0 . 0167 ( 30 mins ) , * * P = 0 . 00301 ( 35 mins ) , * * P = 0 . 00269 ( 40 mins ) , * * P = 0 . 00215 ( 45 mins ) , * * P = 0 . 00129 ( 50mins ) and * * P = 0 . 00141 ( 45mins ) using unpaired 2 - tailed Student ' s t tests .

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

Representative PET, MRI and fused PET/MRI images of the thoracic region of a mouse that received the PDE10A radioligand [18F]-AQ28A. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake (SUV) value of [18F]-AQ28A in BAT and skeletal muscle throughout the dynamic scan was calculated and compared for each animal (n = 5). * P = 0.0488 (20 mins), * P = 0.0236 (25 mins), * P = 0.0167 (30 mins), ** P = 0.00301 (35 mins), ** P = 0.00269 (40 mins), ** P = 0.00215 (45 mins), ** P = 0.00129 (50mins) and ** P = 0.00141 (45mins) using unpaired 2-tailed Student's t tests.


A . Representative PET images of a sagittal view of the thoracic region of fasted lean mice treated with either MP - 10 ( 30mg / kg ) ( n = 3 ) or vehicle ( n = 4 ) prior to receiving [ 18F ] - FDG . Interscapular brown adipose tissue ( BAT ) in each image is highlighted by the crosshairs . The mean standardized uptake value ( SUV ) of [ 18F ] - FDG in BAT after both treatments was calculated for each group . * * P = 0 . 0013 using unpaired 2 - tailed Student ' s t test .

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

A. Representative PET images of a sagittal view of the thoracic region of fasted lean mice treated with either MP-10 (30mg/kg) (n = 3) or vehicle (n = 4) prior to receiving [18F]-FDG. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake value (SUV) of [18F]-FDG in BAT after both treatments was calculated for each group. ** P = 0.0013 using unpaired 2-tailed Student's t test.


B . The percentage ( % ) change in core body temperature and associated area under the curve ( AUC ) of mice treated with MP - 10 ( 30mg / kg ) ( n = 4 ) or vehicle ( n = 4 ) prior to a cold challenge study for 4 hours at 8oC . * P = 0 . 0108 ( 1st hour ) , * P = 0 . 0218 ( 2nd hour ) , * P = 0 . 0121 ( 3rd hour ) , * P = 0 . 0168 ( 4th hour ) and * P = 0 . 0264 ( AUC ) using unpaired 2 - tailed Student ' s t tests .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The percentage (%) change in core body temperature and associated area under the curve (AUC) of mice treated with MP-10 (30mg/kg) (n = 4) or vehicle (n=4) prior to a cold challenge study for 4 hours at 8oC. * P = 0.0108 (1st hour), * P = 0.0218 (2nd hour), * P = 0.0121 (3rd hour), * P = 0.0168 (4th hour) and * P = 0.0264 (AUC) using unpaired 2-tailed Student's t tests.


C . RT - qPCR analysis of relative mRNA expression of Pde10a in BAT , peri - ovarian visceral white adipose tissue ( VAT ) and inguinal subcutaneous white adipose tissue ( SAT ) of lean mice ( n = 5 ) . * * P = 0 . 0063 ( SAT vs VAT ) , * * P = 0 . 0011 ( SAT vs BAT ) and * * P = 0 . 0037 ( VAT vs BAT ) using unpaired 2 - tailed Student ' s t test .

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

C. RT-qPCR analysis of relative mRNA expression of Pde10a in BAT, peri-ovarian visceral white adipose tissue (VAT) and inguinal subcutaneous white adipose tissue (SAT) of lean mice (n = 5). ** P = 0.0063 (SAT vs VAT), ** P = 0.0011 (SAT vs BAT) and ** P = 0.0037 (VAT vs BAT) using unpaired 2-tailed Student's t test.


D Cyclic GMP ( cGMP ) and cyclic AMP ( cGMP ) concentrations normalized to protein concentration in SAT , VAT and BAT 30 minutes after treatment of lean mice with MP - 10 ( 30 mg / kg ) ( n = 6 ) or vehicle control ( n = 6 ) . * * P = 0 . 0085 ( cGMP in VAT ) , * * P = 0 . 0011 ( cGMP in BAT ) and * P = 0 . 04 ( cAMP in VAT ) using unpaired 2 - tailed Student ' s t tests .

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

D Cyclic GMP (cGMP) and cyclic AMP (cGMP) concentrations normalized to protein concentration in SAT, VAT and BAT 30 minutes after treatment of lean mice with MP-10 (30 mg/kg) (n=6) or vehicle control (n = 6). ** P = 0.0085 (cGMP in VAT), ** P = 0.0011 (cGMP in BAT) and * P = 0.04 (cAMP in VAT) using unpaired 2-tailed Student's t tests.


E RT - qPCR analysis of relative mRNA expression of thermogenic genes in SAT , VAT and BAT after treatment of lean mice treated with MP - 10 ( 30 mg / kg ) ( n = 7 ) or vehicle control ( n = 6 ) . * * * * P < 0 . 0001 ( Pgc1alpha in VAT ) , * * P = 0 . 0012 ( Ucp1 in VAT ) , * * P = 0 . 0071 ( Pgc1alpha in BAT ) and * P = 0 . 0486 ( Ucp1 in BAT ) using unpaired 2 - tailed Student ' s t tests .

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

E RT-qPCR analysis of relative mRNA expression of thermogenic genes in SAT, VAT and BAT after treatment of lean mice treated with MP-10 (30 mg/kg) (n=7) or vehicle control (n = 6). **** P < 0.0001 (Pgc1alpha in VAT), ** P = 0.0012 (Ucp1 in VAT), ** P = 0.0071 (Pgc1alpha in BAT) and * P = 0.0486 (Ucp1 in BAT) using unpaired 2-tailed Student's t tests.


Representative fused PET / MRI images of the transverse view of the thoracic region of lean ( n = 5 ) , diet induced obese ( DIO ) ( n = 4 ) and leptin deficient ( ob / ob ) ( n = 5 ) mice that received the PDE10A radioligand [ 18F ] - AQ28A . Interscapular brown adipose tissue ( BAT ) in each image is highlighted by the crosshairs . The mean standardized uptake ( SUV ) value of [ 18F ] - AQ28A in BAT throughout the dynamic scan was calculated with associated area under the curve ( AUC ) . * P = 0 . 0128 ( lean vs DIO ) and * * P = 0 . 0031 ( lean vs ob / ob ) using unpaired 2 - tailed Student ' s t test .

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

Representative fused PET/MRI images of the transverse view of the thoracic region of lean (n = 5), diet induced obese (DIO) (n = 4) and leptin deficient (ob/ob) (n = 5) mice that received the PDE10A radioligand [18F]-AQ28A. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake (SUV) value of [18F]-AQ28A in BAT throughout the dynamic scan was calculated with associated area under the curve (AUC). * P = 0.0128 (lean vs DIO) and ** P = 0.0031 (lean vs ob/ob) using unpaired 2-tailed Student's t test.


A , B The percentage ( % ) change in body weight and cumulative food intake of diet induced obese ( DIO ) mice in response to daily treatment with MP - 10 ( 10mg / kg ) or vehicle for a week ( n = 4 per group ) . * P = 0 . 0264 using ( day 4 ) , * * * P = 0 . 0002 ( day 5 ) , * * * P < 0 . 0001 ( day 6 ) and * * * P < 0 . 0001 ( day 7 ) using Two - Way analysis of variance ( ANOVA ) with Sidak ' s post - hoc test .

O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B The percentage (%) change in body weight and cumulative food intake of diet induced obese (DIO) mice in response to daily treatment with MP-10 (10mg/kg) or vehicle for a week (n= 4 per group). * P = 0.0264 using (day 4), *** P = 0.0002 (day 5), *** P < 0.0001 (day 6) and *** P < 0.0001 (day 7) using Two-Way analysis of variance (ANOVA) with Sidak's post-hoc test.


C . Upon completion of the chronic feeding study , an insulin tolerance test was performed on mice with single treatment of 0 . 75 U / kg insulin ( n = 4 per group ) . * P = 0 . 0246 and * P = 0 . 0305 using unpaired 2 - tailed Student ' s t tests .

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

C. Upon completion of the chronic feeding study, an insulin tolerance test was performed on mice with single treatment of 0.75 U/kg insulin (n = 4 per group). * P = 0.0246 and * P = 0.0305 using unpaired 2-tailed Student's t tests.


O O 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 B-ORGANISM I-ORGANISM O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E, F Upon completion of the chronic feeding study, resting energy expenditure (REE), respiratory quotient (RQ) and locomotor activity of DIO mice treated with MP-10 (30mg/kg) compared to vehicle control were measured in metabolic cage experiments (n = 4 per group). * P = 0.0239 (02:00, RQ), * P = 0.0424 (02:30, RQ), * P = 0.0420 (03:00, RQ), * P = 0.0230 (03:30, RQ), * P = 0.0213 (04:00, RQ), ** P = 0.0116 (00:00, REE), ** P = 0.00823 (00:30, REE), ** P = 0.00969 (01:00, REE), ** P = 0.00274 (01:30, REE), ** P = 0.00323 (02:00, REE), ** P = 0.000441 (02:30, RE), ** P = 0.00390 (03:00, REE), ** P = 0.000211 (03:30, REE), ** P = 0.00216 (04:00, REE) and ** P = 0.0185 (04:30, REE) using unpaired 2-tailed Student's t tests.


G . RT - qPCR analysis of relative mRNA expression of candidate genes in inguinal subcutaneous white adipose tissue ( SAT ) , periovarian visceral white adipose tissue ( VAT ) and interscapularbrown adipose tissue ( BAT ) ( n = 8 per group ) * * * P = 0 . 0004 ( Pgc1alpha VAT ) , * * P = 0 . 0036 ( Ucp1 VAT ) , * * P = 0 . 0068 ( Cidea VAT ) , * P = 0 . 0411 ( Tmem26 VAT ) and * * P = 0 . 0070 ( Pgc1alpha BAT ) using unpaired 2 - tailed Student ' s t tests .

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

G. RT-qPCR analysis of relative mRNA expression of candidate genes in inguinal subcutaneous white adipose tissue (SAT), periovarian visceral white adipose tissue (VAT) and interscapularbrown adipose tissue (BAT) (n = 8 per group) *** P = 0.0004 (Pgc1alpha VAT), ** P = 0.0036 (Ucp1 VAT), ** P = 0.0068 (Cidea VAT), * P = 0.0411 (Tmem26 VAT) and ** P = 0.0070 (Pgc1alpha BAT) using unpaired 2-tailed Student's t tests.


H . Representative sections of inguinal SAT , peri - ovarian VAT and interscapular BAT immunostained for UCP1 of mice chronically treated with MP - 10 ( 10 mg / kg ) or vehicle control . Scale bars , 100µm .

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

H. Representative sections of inguinal SAT, peri-ovarian VAT and interscapular BAT immunostained for UCP1 of mice chronically treated with MP-10 (10 mg/kg) or vehicle control. Scale bars, 100µm.


Representative PET image of the upper body of a human subject who received the PDE10A radioligand [ 18F ] - MNI - 1869 . Supraclavicular brown adipose tissue ( BAT ) is highlighted by the crosshairs . The mean standardized uptake value ( SUV ) of [ 18F ] - MNI - 1869 in supraclavicular BAT and skeletal muscle was calculated in human subjects ( n = 5 ) . * * P = 0 . 00210 ( 5 mins ) , * * * * P < 0 . 0001 ( 15 mins ) , * * * * P < 0 . 0001 ( 30 mins ) , * * * * P < 0 . 0001 ( 50 mins ) and * * P = 0 . 00462 ( 75 mins ) using unpaired 2 - tailed Student ' s t tests .

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

Representative PET image of the upper body of a human subject who received the PDE10A radioligand [18F]-MNI-1869. Supraclavicular brown adipose tissue (BAT) is highlighted by the crosshairs. The mean standardized uptake value (SUV) of [18F]-MNI-1869 in supraclavicular BAT and skeletal muscle was calculated in human subjects (n = 5). ** P = 0.00210 (5 mins), **** P < 0.0001 (15 mins), **** P < 0.0001 (30 mins), **** P < 0.0001 (50 mins) and ** P = 0.00462 (75 mins) using unpaired 2-tailed Student's t tests.