Figure 5B Bright field images of hFI , hCI , hPI and hPI + MVF . Scale bar : 100 µm . C Representative immunohistochemical stainings of insulin and CD31 in hFI , hCI , hPI and hPI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 100 µm . D - G Quantitative analysis of insulin - ( D ) , glucagon - ( E ) , somatostatin - ( F ) and CD31 - positive cells ( G ) in hFI , hCI , hPI and hPI + MVF in % of all islet or islet organoid cells ( n = 20 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . hFI ; # P < 0 . 05 vs . hCI ; + P < 0 . 05 vs . hPI . H Representative bright field images of angiogenic sprouting of hFI , hCI , hPI and hPI + MVF on day 3 of the sprouting assay . Scale bar : 150 µm . I Quantitative analysis of sprouting area of hFI , hCI , hPI and hPI + MVF in % of initial size ( day 0 ) ( n = 10 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . hFI ; # P < 0 . 05 vs . hCI ; + P < 0 . 05 vs . hPI . J Quantitative analysis of C - peptide secretion ( pg / mL ) ( normalized to DNA concentration ) of hFI and hPI + MVF exposed to 1 . 1 mM and 16 . 5 mM glucose ( n = 3 each ) . Mean ± SD .

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

Figure 5B Bright field images of hFI, hCI, hPI and hPI+MVF. Scale bar: 100 µm.C Representative immunohistochemical stainings of insulin and CD31 in hFI, hCI, hPI and hPI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 100 µm.D-G Quantitative analysis of insulin-(D), glucagon-(E), somatostatin-(F) and CD31-positive cells (G) in hFI, hCI, hPI and hPI+MVF in % of all islet or islet organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. hFI; #P < 0.05 vs. hCI; +P < 0.05 vs. hPI.H Representative bright field images of angiogenic sprouting of hFI, hCI, hPI and hPI+MVF on day 3 of the sprouting assay. Scale bar: 150 µm.I Quantitative analysis of sprouting area of hFI, hCI, hPI and hPI+MVF in % of initial size (day 0) (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. hFI; #P < 0.05 vs. hCI; +P < 0.05 vs. hPI.J Quantitative analysis of C-peptide secretion (pg/mL) (normalized to DNA concentration) of hFI and hPI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD.


Figure 6B Observation window of a dorsal skinfold chamber with 8 neutral red - stained transplanted FI on day 0 . Scale bar : 5 mm . C Take rate ( % of transplanted islets ) on day 14 after islet transplantation into the dorsal skinfold chamber ( n = 8 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . + P < 0 . 05 vs . PI . D Representative intravital fluorescent microscopic images of FI , CI , PI and PI + MVF within the dorsal skinfold chamber on day 0 and 14 . FITC - labeled dextran is used for the visualization of blood - perfused microvessels . Grafts are marked by broken lines . Scale bar : 150 μm . E , F Quantitative analysis of the functional capillary density ( cm / cm - 2 ) ( E ) and revascularized area ( mm2 ) ( F ) of transplanted FI , CI , PI and PI + MVF on day 0 , 3 , 6 , 10 and 14 ( n = 8 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI . G Representative intravital fluorescent microscopic images of FI , CI , PI and PI + MVF within the dorsal skinfold chamber on day 14 . Rhodamine 6G is used to visualize blood perfused vital endocrine cells ( bright signals ) . Scale bar : 150 µm . H Quantitative analysis of the rhodamine 6G - positive area ( % of islet size ) within transplanted FI , CI , PI and PI + MVF on day 0 , 3 , 6 , 10 and 14 . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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

Figure 6B Observation window of a dorsal skinfold chamber with 8 neutral red-stained transplanted FI on day 0. Scale bar: 5 mm.C Take rate (% of transplanted islets) on day 14 after islet transplantation into the dorsal skinfold chamber (n = 8 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. +P < 0.05 vs. PI.D Representative intravital fluorescent microscopic images of FI, CI, PI and PI+MVF within the dorsal skinfold chamber on day 0 and 14. FITC-labeled dextran is used for the visualization of blood-perfused microvessels. Grafts are marked by broken lines. Scale bar: 150 μm.E, F Quantitative analysis of the functional capillary density (cm/cm-2) (E) and revascularized area (mm2) (F) of transplanted FI, CI, PI and PI+MVF on day 0, 3, 6, 10 and 14 (n = 8 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.G Representative intravital fluorescent microscopic images of FI, CI, PI and PI+MVF within the dorsal skinfold chamber on day 14. Rhodamine 6G is used to visualize blood perfused vital endocrine cells (bright signals). Scale bar: 150 µm.H Quantitative analysis of the rhodamine 6G-positive area (% of islet size) within transplanted FI, CI, PI and PI+MVF on day 0, 3, 6, 10 and 14. Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


Figure 7A Representative immunofluorescent stainings of insulin / glucagon , insulin / somatostatin and insulin / CD31 in FI , CI , PI and PI + MVF on day 14 after transplantation . Cell nuclei are stained with Hoechst 33342 ( blue ) . Arrows mark intra - islet endothelial cells . Scale bar : 70 µm . B - E Quantitative analysis of insulin - ( B ) , glucagon - ( C ) , somatostatin - ( D ) and intra - islet CD31 - positive cells ( E ) within FI , CI , PI and PI + MVF on day 14 after transplantation ( n = 10 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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Figure 7A Representative immunofluorescent stainings of insulin / glucagon, insulin / somatostatin and insulin / CD31 in FI, CI, PI and PI+MVF on day 14 after transplantation. Cell nuclei are stained with Hoechst 33342 (blue). Arrows mark intra-islet endothelial cells. Scale bar: 70 µm. B-E Quantitative analysis of insulin- (B), glucagon- (C), somatostatin- (D) and intra-islet CD31-positive cells (E) within FI, CI, PI and PI+MVF on day 14 after transplantation (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


Figure 8B Quantitative analysis of the body weight ( g ) of nondiabetic and diabetic mice transplanted with FI or PI + MVF from day - 8 to day 28 ( n = 7 each ) . Mean ± SD . C AUC of the body weight of nondiabetic and diabetic mice transplanted with FI or PI + MVF from day 0 to day 28 ( n = 7 each ) . Mean ± SD . D Quantitative analysis of blood glucose level ( mg / dL ) of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls from day - 8 to day 28 ( n = 7 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . PI + MVF . E AUC of the blood glucose levels of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls from day 0 to day 28 ( n = 7 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . PI + MVF . F Quantitative analysis of blood glucose level ( mg / dL ) according to the IPGTT of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls to the indicated time points ( n = 5 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI . G AUC of IPGTT of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls ( n = 5 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . PI + MVF . H Plasma insulin level ( µU / mL ) of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls at 180 min after IPGTT ( n = 4 each ) . Mean ± SD . I Representative immunofluorescent stainings of insulin / CD31 in FI and PI + MVF on day 28 after islet transplantation . Scale bar : 50 µm . J Quantitative analysis of insulin - ( β - cells ) , glucagon - ( α - cells ) , somatostatin - ( δ - cells ) and CD31 - positive ( endothelial ) cells within transplanted FI and PI + MVF on day 28 after islet transplantation . Data are expressed in % of total islet cells ( n = 25 ) . Mean ± SD . K Quantitative analysis of islet cells in transplanted FI and PI + MVF per HPF ( n = 70 each ) . Mean ± SD . Unpaired Student ' s t - test was used for statistical analysis . * P < 0 . 05 vs . FI .

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

Figure 8B Quantitative analysis of the body weight (g) of nondiabetic and diabetic mice transplanted with FI or PI+MVF from day -8 to day 28 (n = 7 each). Mean ± SD.C AUC of the body weight of nondiabetic and diabetic mice transplanted with FI or PI+MVF from day 0 to day 28 (n = 7 each). Mean ± SD.D Quantitative analysis of blood glucose level (mg/dL) of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls from day -8 to day 28 (n = 7 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.E AUC of the blood glucose levels of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls from day 0 to day 28 (n = 7 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.F Quantitative analysis of blood glucose level (mg/dL) according to the IPGTT of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls to the indicated time points (n = 5 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI.G AUC of IPGTT of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls (n = 5 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.H Plasma insulin level (µU/mL) of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls at 180 min after IPGTT (n = 4 each). Mean ± SD.I Representative immunofluorescent stainings of insulin / CD31 in FI and PI+MVF on day 28 after islet transplantation. Scale bar: 50 µm.J Quantitative analysis of insulin- (β-cells), glucagon- (α-cells), somatostatin- (δ-cells) and CD31-positive (endothelial) cells within transplanted FI and PI+MVF on day 28 after islet transplantation. Data are expressed in % of total islet cells (n = 25). Mean ± SD.K Quantitative analysis of islet cells in transplanted FI and PI+MVF per HPF (n = 70 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. FI.


Figure 1A . NS1 dimer with one subunit in grey and the other colored by domain ( green , β - roll ; orange , wing with magenta connector subdomain ; blue , central β - ladder ) . N - linked glycosylation sites are shown in sticks , and the glycans are shown in spheres . B . Topology diagram for the NS1 monomer , colored as the pattern in ( A ) . Glycosylation sites are indicated with red hexagons and disulfide bonds with yellow circles . C . Side views of the NS1 dimer from the wing ( left ) and the end of the β - ladder ( right ) . The β - roll ( green ) , connector subdomain ( orange ) and the intertwined loop ( yellow ) of the wing domain form a discontinuous protrusion on one face of the β - ladder with the spaghetti loop ( cyan ) on the other face . The wing domain is omitted from the left image for clarity .

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Figure 1A. NS1 dimer with one subunit in grey and the other colored by domain (green, β-roll; orange, wing with magenta connector subdomain; blue, central β-ladder). N-linked glycosylation sites are shown in sticks, and the glycans are shown in spheres.B. Topology diagram for the NS1 monomer, colored as the pattern in (A). Glycosylation sites are indicated with red hexagons and disulfide bonds with yellow circles.C. Side views of the NS1 dimer from the wing (left) and the end of the β-ladder (right). The β-roll (green), connector subdomain (orange) and the intertwined loop (yellow) of the wing domain form a discontinuous protrusion on one face of the β-ladder with the spaghetti loop (cyan) on the other face. The wing domain is omitted from the left image for clarity.


Figure 2The ZIKV NS1 hexamer is modeled by using the full - length DENV2 NS1 structure ( PDB code : 4O6B ) ( Akey et al , 2014 ) . Each monomer is colored by domains ( green , β - roll ; orange , wing ; magenta , connector subdomain ; blue , central β - ladder ) . The intertwined loop " spike " of ZIKV NS1 contributing to membrane association is colored in red and indicated by dotted lines .

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Figure 2The ZIKV NS1 hexamer is modeled by using the full-length DENV2 NS1 structure (PDB code: 4O6B) (Akey et al, 2014). Each monomer is colored by domains (green, β-roll; orange, wing; magenta, connector subdomain; blue, central β-ladder). The intertwined loop "spike" of ZIKV NS1 contributing to membrane association is colored in red and indicated by dotted lines.


Figure 3ABC . Electrostatic surface views of NS1 from DENV2 ( PDB : 4O6B , A ) and WNV ( PDB : 4O6C , B ) ( Akey et al , 2014 ) , as well as ZIKV © , showing diverse characteristics on certain regions . Surfaces are colored by electrostatic potential at neutral pH from - 2 kT ( red ) to + 2 kT ( blue ) using PyMOL ( http : / / pymol . org / ) .

Figure 3ABC. Electrostatic surface views of NS1 from DENV2 (PDB: 4O6B, A) and WNV (PDB: 4O6C, B) (Akey et al, 2014), as well as ZIKV©, showing diverse characteristics on certain regions. Surfaces are colored by electrostatic potential at neutral pH from -2 kT (red) to +2 kT (blue) using PyMOL (http://pymol.org/).


Figure 4A . The NS1 surface is colored using the ConSurf server ( Ashkenazy et al , 2016 ) according to sequence conservation from the most conserved ( dark magenta ) to the most divergent ( dark cyan ) based on an alignment of NS1 sequences from 73 flaviviruses . B . Models of intertwined loop " spike " interacting with the cellular membrane . The disordered " spike " regions are indicated by dash lines in DENV2 and WNV NS1 structures , which consist of hydrophobic or positively charged amino acids .

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Figure 4A. The NS1 surface is colored using the ConSurf server (Ashkenazy et al, 2016) according to sequence conservation from the most conserved (dark magenta) to the most divergent (dark cyan) based on an alignment of NS1 sequences from 73 flaviviruses.B. Models of intertwined loop "spike" interacting with the cellular membrane. The disordered "spike" regions are indicated by dash lines in DENV2 and WNV NS1 structures, which consist of hydrophobic or positively charged amino acids.


Figure 5A . B - factor putty representation of the intertwined loop structure . Shades of blue and a narrower tube indicate regions with lower B - factors , whereas orange to red and a wider tube indicate regions with higher B - factors . The figure was prepared using PyMOL ( http : / / pymol . org / ) . B . The 2Fo - Fc electron density map for the intertwined loop ( yellow ) " spike " region residues contoured at 1 . 0 sigma is represented in blue . C . Detailed interactions of the highly conserved residues W115 and W118 with the neighboring residues to stabilize the conformation of the intertwined loop . W115 forms a hydrogen - bonding interaction with E74 from α2 helix . W118 inserts into the hydrophobic interspace between α1 and α2 helix , formed by P36 , L42 , V71 , L75 and I78 , and W118 also interact with P36 and D37 by hydrogen - bonding interactions .

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Figure 5A. B-factor putty representation of the intertwined loop structure. Shades of blue and a narrower tube indicate regions with lower B-factors, whereas orange to red and a wider tube indicate regions with higher B-factors. The figure was prepared using PyMOL (http://pymol.org/).B. The 2Fo-Fc electron density map for the intertwined loop (yellow) "spike" region residues contoured at 1.0 sigma is represented in blue.C. Detailed interactions of the highly conserved residues W115 and W118 with the neighboring residues to stabilize the conformation of the intertwined loop. W115 forms a hydrogen-bonding interaction with E74 from α2 helix. W118 inserts into the hydrophobic interspace between α1 and α2 helix, formed by P36, L42, V71, L75 and I78, and W118 also interact with P36 and D37 by hydrogen-bonding interactions.


Figure 1Immunofluorescence staining of RANKL during in vitro adipogenesis of BMSCs from AdipoqCre ; ranklfl / fl mice ( Rankl - / - ) and ranklfl / fl mice ( Rankl + / + ) . Scale bar = 50 μm .

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Figure 1Immunofluorescence staining of RANKL during in vitro adipogenesis of BMSCs from AdipoqCre; ranklfl/fl mice (Rankl-/-) and ranklfl/fl mice (Rankl+/+). Scale bar=50 μm.


Figure 2Micro - CT and statistical analyses of BMD , BV / TV , Tb . N and Tb . Sp in trabecular bone from AdipoqCre ; ranklfl / fl ( Rankl - / - ) and ranklfl / fl ( Rankl + / + ) mice at 4 weeks , 8 weeks and 16 weeks ( n = 5 independent biological replicates ) . Scale bar = 1 mm . Data were compared using an unpaired t - test ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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Figure 2Micro-CT and statistical analyses of BMD, BV/TV, Tb.N and Tb.Sp in trabecular bone from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 4 weeks, 8 weeks and 16 weeks (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t-test (** indicates P < 0.01), error bars are standard deviations.


Figure 3Representative images of TRAP staining of femur from AdipoqCre ; ranklfl / fl ( Rankl - / - ) and ranklfl / fl ( Rankl + / + ) mice at 8 weeks . Scale bar = 200 μm . Panel a1 and a4 indicate trabecular bone , a2 and a5 stand for growth plate , a3 and a6 denote cortical bone . Immunofluorescence staining of osteocalcin from 8 - week - old mice ( n = 6 mice in Rankl - / - group and 4 mice in Rankl + / + group ) . Scale bar = 50 μm . TB : trabecular bone . Statistical analyses of Ocn + cells ( n = 6 independent microscopic vision fields from random mice samples ) . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations . Calcein staining of trabecular bone at 8 weeks . Scale bar = 50 μm . TB : trabecular bone . Images are representative of 6 independent biological replicates in Rankl - / - and 5 replicates in Rankl + / + group . Mineral apposition rate ( MAR ) of trabecular bone at 8 weeks . ( n = 6 mice in Rankl - / - group and 5 mice in Rankl + / + group ) . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations . Serum CTX - 1 , OCN and bone marrow RANKL levels from Rankl - / - and Rankl + / + mice at 4 weeks ( n = 5 and 5 ) , 8 weeks ( n = 5 and 6 ) and 16 weeks ( n = 5 and 5 ) . Data were compared using an unpaired t ‐ test ( * indicates P < 0 . 05 , * * indicates P < 0 . 01 ) , error bars are standard deviations . .

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

Figure 3Representative images of TRAP staining of femur from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 8 weeks. Scale bar=200 μm. Panel a1 and a4 indicate trabecular bone, a2 and a5 stand for growth plate, a3 and a6 denote cortical bone.Immunofluorescence staining of osteocalcin from 8-week-old mice (n=6 mice in Rankl-/- group and 4 mice in Rankl+/+ group). Scale bar=50 μm. TB: trabecular bone. Statistical analyses of Ocn+ cells (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.Calcein staining of trabecular bone at 8 weeks. Scale bar=50 μm. TB: trabecular bone. Images are representative of 6 independent biological replicates in Rankl-/- and 5 replicates in Rankl+/+ group. Mineral apposition rate (MAR) of trabecular bone at 8 weeks.(n=6 mice in Rankl-/- group and 5 mice in Rankl+/+ group). Data were compared using an unpaired t‐test ( ** indicates P < 0.01), error bars are standard deviations.Serum CTX-1, OCN and bone marrow RANKL levels from Rankl-/- and Rankl+/+ mice at 4 weeks (n=5 and 5), 8 weeks (n=5 and 6) and 16 weeks (n=5 and 5). Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations..


Figure 4Micro - CT analyses of trabecular bone from Grounded and Tail suspended model ( n = 5 independent biological replicates ) . Scale bar = 1 mm . Data were compared using an unpaired t ‐ test ( * indicates P < 0 . 05 , * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Figure 4Micro-CT analyses of trabecular bone from Grounded and Tail suspended model (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations.


Figure 5Micro - CT analyses of sham and OVX model of AdipoqCre ; ranklfl / fl ( Rankl - / - ) and ranklfl / fl ( Rankl + / + ) mice ( n = 5 independent biological replicates ) . Scale bar = 1 mm . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations . TRAP staining and statistical analyses of numbers of osteoclasts in Sham and OVX model ( Images are representative of 5 independent biological replicates in ( C ) , and n = 5 independent microscopic vision fields in ( D ) ) . Scale bar = 100 µm . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations . Immunofluorescence staining of OCN and statistical analyses of the number of osteoblasts ( Images are representative of 5 independent biological replicates in ( E ) , and n = 5 independent microscopic vision fields in ( F ) ) . Scale bar = 50 µm . TB : trabecular bone . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Figure 5Micro-CT analyses of sham and OVX model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.TRAP staining and statistical analyses of numbers of osteoclasts in Sham and OVX model (Images are representative of 5 independent biological replicates in (C), and n=5 independent microscopic vision fields in (D)). Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.Immunofluorescence staining of OCN and statistical analyses of the number of osteoblasts (Images are representative of 5 independent biological replicates in (E), and n=5 independent microscopic vision fields in (F)). Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


Figure 6Three - dimensional CT analyses of BMD , BV / TV , Tb . N , and Tb . Sp in trabecular bone from Vehicle ( n = 5 independent biological replicates ) and ROS ( n = 6 independent biological replicates ) model of AdipoqCre ; ranklfl / fl ( Rankl - / - ) and ranklfl / fl ( Rankl + / + ) mice . Scale bar = 1 mm . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations . TRAP staining and statistical analyses of numbers of osteoclasts in the trabecular bone from Vehicle ( n = 6 and 5 independent biological replicates ) and ROS ( n = 6 and 6 independent biological replicates ) treatment . Scale bar = 100 µm . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations . n = 5 independent microscopic vision fields in ( D ) . OCN staining and statistical analyses of the number of osteoblasts in femurs from Vehicle ( n = 6 and 5 independent biological replicates ) and ROS ( n = 6 and 6 independent biological replicates ) treatment . Scale bar = 50 µm . TB : trabecular bone . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations . n = 5 independent microscopic vision fields in ( F ) .

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

Figure 6Three-dimensional CT analyses of BMD, BV/TV, Tb.N, and Tb.Sp in trabecular bone from Vehicle (n=5 independent biological replicates) and ROS (n=6 independent biological replicates) model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice. Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.  TRAP staining and statistical analyses of numbers of osteoclasts in the trabecular bone from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (D).OCN staining and statistical analyses of the number of osteoblasts in femurs from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (F).


Figure 1A - C Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( A ) body weight and ( B , C ) body composition . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

Figure 1A-C Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A) body weight and (B, C) body composition. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 2A - G Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( A , B ) energy expenditure . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - G Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( C ) respiratory exchange ratio ( RER ) . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - G Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( D ) locomotor activity . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - G Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( E ) cumulative food intake , ( F ) meal number and ( G ) meal size . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

Figure 2A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A, B) energy expenditure. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (C) respiratory exchange ratio (RER). Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (D) locomotor activity. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (E) cumulative food intake, (F) meal number and (G) meal size. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 3A , B Treatment - induced changes in hypothalamic gene expression in DIO mice treated for 2 ( A ) or 5 ( B ) days with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Compounds were administered by daily subcutaneous injections , and the last injection was provided 2 h prior to tissues sampling . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , § P < 0 , 001 , comparison between treatment and vehicle control . # P < 0 . 05 , # # P < 0 . 01 , # # # P < 0 . 001 , comparison to liraglutide . + P < 0 . 05 , comparison to RM - 493 .

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

Figure 3A, B Treatment-induced changes in hypothalamic gene expression in DIO mice treated for 2 (A) or 5 (B) days with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Compounds were administered by daily subcutaneous injections, and the last injection was provided 2 h prior to tissues sampling. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, §P < 0,001, comparison between treatment and vehicle control. #P < 0.05, ##P < 0.01, ###P < 0.001, comparison to liraglutide. +P < 0.05, comparison to RM-493.


Figure 4A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( A ) Fasted blood glucose levels . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( B , C ) glucose tolerance . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( D ) fasted insulin levels , . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( E , F ) glucose - induced insulin secretion . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( G ) HOMA - IR . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( H ) insulin sensitivity . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( I ) hepatic phosphorylation of AKT ( p - AKTSer473 ) were analyzed . Compounds were administered by daily subcutaneous injections . To assess insulin - stimulated p - AKTSer473 , insulin ( n = 5 ) or saline ( n = 3 ) was injected 10 min prior to liver sampling . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

Figure 4A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (A) Fasted blood glucose levels. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered).(B, C) glucose tolerance. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (D) fasted insulin levels,. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (E, F) glucose-induced insulin secretion. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (G) HOMA-IR. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (H) insulin sensitivity. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (I) hepatic phosphorylation of AKT (p-AKTSer473) were analyzed. Compounds were administered by daily subcutaneous injections. To assess insulin-stimulated p-AKTSer473, insulin (n = 5) or saline (n = 3) was injected 10 min prior to liver sampling. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 5A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( A ) Plasma lipoprotein fractions . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( B ) liver cholesterol levels . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( C ) expression of genes implicated in hepatic cholesterol and lipoprotein uptake . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( D ) expression of genes implicated in cholesterol and bile acid metabolism and excretion were analyzed . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

Figure 5A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (A) Plasma lipoprotein fractions. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (B) liver cholesterol levels. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (C) expression of genes implicated in hepatic cholesterol and lipoprotein uptake. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (D) expression of genes implicated in cholesterol and bile acid metabolism and excretion were analyzed. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 6A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( A ) body weight . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( B ) cumulative food intake . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( C , D ) body composition . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( E , F ) glucose tolerance . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

Figure 6A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A) body weight. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (B) cumulative food intake. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (C, D) body composition. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (E, F) glucose tolerance. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 1 ( A ) Size exclusion chromatography profile of NS1 pull - down experiments showing a clear shift after incubation in complete or heat - inactivated human serum ( solid and dotted black lines , respectively ) from the same healthy donor compared to the NS1 protein alone ( blue line ) . NS1 protein interaction partners were identified by SDS - PAGE and N - terminal sequencing as the Apolipoprotein B scaffold of the low - density lipoproteins ( LDL ) in the first SEC elution peak , and the ApoA - I protein scaffold of the high - density lipoproteins ( HDL ) in the second elution peak . ( B ) Biolayer interferometry ( BLI ) profiles corresponding to the binding of NS1 at various concentrations respective to human HDL ( left panel ) and human LDL ( central panel ) particles . The concentration - dependence of the steady - state signal corresponding to the binding of NS1 to HDL ( black dots ) and LDL ( white dots ) is shown on the right - hand side panel . The measurements were replicated at least three times using novel biosensors and samples . Data points and error bars correspond to the mean ± SD . ( C ) Typical sedimentation coefficient distribution of NS1 or human HDL alone or pre - incubated together ( mixture of NS1 and HDL at a 1 : 1 or 5 : 1 mass ratio ) monitored using an interferometric detector . Peaks were integrated for all the detectors ( interference and absorbance at 280nm ) . The calculated stoichiometries are indicated for each peak . Solutions were equilibrated at 20 ° C for 2h before sedimentation velocity analysis .

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

Figure 1(A) Size exclusion chromatography profile of NS1 pull-down experiments showing a clear shift after incubation in complete or heat-inactivated human serum (solid and dotted black lines, respectively) from the same healthy donor compared to the NS1 protein alone (blue line). NS1 protein interaction partners were identified by SDS-PAGE and N-terminal sequencing as the Apolipoprotein B scaffold of the low-density lipoproteins (LDL) in the first SEC elution peak, and the ApoA-I protein scaffold of the high-density lipoproteins (HDL) in the second elution peak.(B) Biolayer interferometry (BLI) profiles corresponding to the binding of NS1 at various concentrations respective to human HDL (left panel) and human LDL (central panel) particles. The concentration-dependence of the steady-state signal corresponding to the binding of NS1 to HDL (black dots) and LDL (white dots) is shown on the right-hand side panel. The measurements were replicated at least three times using novel biosensors and samples. Data points and error bars correspond to the mean ± SD.(C) Typical sedimentation coefficient distribution of NS1 or human HDL alone or pre-incubated together (mixture of NS1 and HDL at a 1:1 or 5:1 mass ratio) monitored using an interferometric detector. Peaks were integrated for all the detectors (interference and absorbance at 280nm). The calculated stoichiometries are indicated for each peak. Solutions were equilibrated at 20°C for 2h before sedimentation velocity analysis.


Figure 2 ( A , B ) Electron microscopy observations from left to right : a representative image , followed by the three most representative classes of ( A ) purified HDL particles and ( B ) NS1 - HDL complexes . White bar : 50 nm , Black bar : 20 nm . ( D ) Differential scanning calorimetry ( DSC ) of NS1 alone ( blue line ) or of an NS1 - HDL mixture at a 2 : 1 molar ratio ( orange ) . Of note , the HDL particles alone did not generate any signal in the temperature range tested . ( E ) Binding inhibition of hexameric NS1 to HDL with anti - ApoA - I polyclonal antibodies ( anti - ApoA - I Ab ) measured by BLI .

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

Figure 2(A, B) Electron microscopy observations from left to right: a representative image, followed by the three most representative classes of (A) purified HDL particles and (B) NS1-HDL complexes. White bar: 50 nm, Black bar: 20 nm.(D) Differential scanning calorimetry (DSC) of NS1 alone (blue line) or of an NS1-HDL mixture at a 2:1 molar ratio (orange). Of note, the HDL particles alone did not generate any signal in the temperature range tested.(E) Binding inhibition of hexameric NS1 to HDL with anti-ApoA-I polyclonal antibodies (anti-ApoA-I Ab) measured by BLI.


Figure 3 ( A - D ) Human primary macrophages were incubated for 24h with the different potential effectors ( NS1 , HDL , mix NS1 - HDL ) or with control suspensions ( PBS buffer , LPS , mix LPS - HDL ) . LPS stimulation was used as a positive control in the presence or absence of HDL and provided values consistent between experiments . Phosphate buffer used in the SEC purification step was used as a negative control . Cell culture supernatants were clarified and tested with a Luminex assay to quantify the amount of ( A ) TNF - α , ( B ) Il - 6 , ( C ) Il - 1ß and ( D ) Il - 10 released in the extracellular medium . Data reported on the graphs correspond to biological replicates of macrophages isolated from four blood donors ( n = 9 for TNF - α , Il - 6 and Il - 1ß , n = 5 for Il - 10 ) . Data represent mean + / - SEM . A Mann - Whitney test was used to assess the statistical significance of differences observed between mean cytokine levels in different cell culture supernatants . Not significant : ns , * p < 0 . 05 , * * * p < 0 . 001 .

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

Figure 3(A-D) Human primary macrophages were incubated for 24h with the different potential effectors (NS1, HDL, mix NS1-HDL) or with control suspensions (PBS buffer, LPS, mix LPS-HDL). LPS stimulation was used as a positive control in the presence or absence of HDL and provided values consistent between experiments. Phosphate buffer used in the SEC purification step was used as a negative control. Cell culture supernatants were clarified and tested with a Luminex assay to quantify the amount of (A) TNF-α, (B) Il-6, (C) Il-1ß and (D) Il-10 released in the extracellular medium. Data reported on the graphs correspond to biological replicates of macrophages isolated from four blood donors (n=9 for TNF-α, Il-6 and Il-1ß, n=5 for Il-10). Data represent mean +/- SEM. A Mann-Whitney test was used to assess the statistical significance of differences observed between mean cytokine levels in different cell culture supernatants. Not significant: ns, * p < 0.05, *** p < 0.001.


Figure 4levels of ( B ) NS1 , ( C ) NS1 - ApoA - I , ( D ) NS1 - ApoE and ( E ) NS1 - ApoB NS1 - ApoA - I and NS1 - ApoB complexes are representative of NS1 - HDL and NS1 - LDL complex species , respectively , while the NS1 - ApoE - positive complexes remain to be fully characterized . Errors bars indicate SEM . levels of ( F ) total cholesterol , ( G ) HDL - cholesterol and ( H ) triglycerides . Errors bars indicate SEM . ( I ) Purified NS1 from different flaviviruses ( yellow fever , YF ; ZIKA ; West Nile , WN ; Japanese encephalitis , JE ; The Native Antigen Company ) were spiked in normal human plasma and NS1 - ApoA - I complexes further detected by ELISA . Data represent the mean values of two technical replicates .

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

Figure 4levels of (B) NS1, (C) NS1-ApoA-I, (D) NS1-ApoE and (E) NS1-ApoB NS1-ApoA-I and NS1-ApoB complexes are representative of NS1-HDL and NS1-LDL complex species, respectively, while the NS1-ApoE-positive complexes remain to be fully characterized. Errors bars indicate SEM.levels of (F) total cholesterol, (G) HDL-cholesterol and (H) triglycerides. Errors bars indicate SEM.(I) Purified NS1 from different flaviviruses (yellow fever, YF; ZIKA; West Nile, WN; Japanese encephalitis, JE; The Native Antigen Company) were spiked in normal human plasma and NS1-ApoA-I complexes further detected by ELISA. Data represent the mean values of two technical replicates.


Figure 1A ) Shear - dilation relations of Tek - Cre : : Lama5 - / - and Lama4 - / - mesenteric resistance arteries and corresponding wild type controls show a reduced response of Tek - Cre : : Lama5 - / - arteries and an enhanced response of Lama4 - / - arteries . Data shown are mean changes in vessel diameter ( Δ µm ) ± s . e . m . from 8 experiments with 16 wild type and 16 KO arteries ; KO and wild type were analysed as pairs in each experiment . * * P < 0 . 01 , paired t - test . The dose - response curves of arteries from wild type , Tek - Cre : : Lama5 - / - ( B ) and Lama4 - / - mice ( C ) and wild type littermates stimulated with methacholine do not show significant differences ( n . s . ) . Data are expressed as percent relaxation of the maximum force developed in presence of 0 . 3 µM U46619 , and are mean values ± s . e . m from 7 experiments with 1 wild type and 1 KO artery in each experiment .

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

Figure 1A) Shear-dilation relations of Tek-Cre::Lama5-/- and Lama4-/- mesenteric resistance arteries and corresponding wild type controls show a reduced response of Tek-Cre::Lama5-/- arteries and an enhanced response of Lama4-/- arteries. Data shown are mean changes in vessel diameter (Δ µm) ± s.e.m. from 8 experiments with 16 wild type and 16 KO arteries; KO and wild type were analysed as pairs in each experiment. **P<0.01, paired t-test.The dose-response curves of arteries from wild type, Tek-Cre::Lama5-/- (B) and Lama4-/- mice (C) and wild type littermates stimulated with methacholine do not show significant differences (n.s.). Data are expressed as percent relaxation of the maximum force developed in presence of 0.3 µM U46619, and are mean values ± s.e.m from 7 experiments with 1 wild type and 1 KO artery in each experiment.


Figure 2A ) Three - dimensional digital reconstructions of optical sections through immunofluorescently stained mesenteric resistance arteries from wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice , and optical sections through vessel walls to show the endothelial and underlying smooth muscle layers . DAPI staining permits identification of endothelial cell nuclei , which lie perpendicular to the nuclei of the smooth muscle cells . Laminin α5 is absent from endothelial basement membranes of Tek - Cre : : Lama5 - / - arteries ( arrow ) but is still present in smooth muscle basement membranes ( asterisk ) , while laminin α4 is still detectable in both endothelial and smooth muscle basement membranes . Lama4 - / - arteries lack laminin α4 in both endothelial ( arrow ) and smooth muscle ( asterisk ) basement membranes , but laminin α5 is still detectable . Scale bars are 10 μm . B ) Scanning electron microscopy images of endothelial cell - denuded mesenteric resistance arteries show a comparable topography of the endothelial basement membrane in wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - arteries . The arrows indicate artificial ruptures due to preparation procedure . Scale bars are 100 nm . C ) Intravital microscopic quantification of mesenteric arteries sizes in vivo show smaller diameters ( - 24 . 3 % ) in Tek - Cre : : Lama5 - / - arteries and larger diameters ( + 27 . 6 % ) in Lama4 - / - arteries compared to wild type controls . Auto - fluorescence of the internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter . Scale bar is 100 µm . Data are means ± s . e . m from a minimum of 4 first order mesenteric arteries imaged at least in 4 mice per genotype . * P < 0 . 05 unpaired t - test .

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

Figure 2A) Three-dimensional digital reconstructions of optical sections through immunofluorescently stained mesenteric resistance arteries from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice, and optical sections through vessel walls to show the endothelial and underlying smooth muscle layers. DAPI staining permits identification of endothelial cell nuclei, which lie perpendicular to the nuclei of the smooth muscle cells. Laminin α5 is absent from endothelial basement membranes of Tek-Cre::Lama5-/- arteries (arrow) but is still present in smooth muscle basement membranes (asterisk), while laminin α4 is still detectable in both endothelial and smooth muscle basement membranes. Lama4-/- arteries lack laminin α4 in both endothelial (arrow) and smooth muscle (asterisk) basement membranes, but laminin α5 is still detectable. Scale bars are 10 μm.B) Scanning electron microscopy images of endothelial cell-denuded mesenteric resistance arteries show a comparable topography of the endothelial basement membrane in wild type, Tek-Cre::Lama5-/- and Lama4-/- arteries. The arrows indicate artificial ruptures due to preparation procedure. Scale bars are 100 nm.C) Intravital microscopic quantification of mesenteric arteries sizes in vivo show smaller diameters (-24.3%) in Tek-Cre::Lama5-/- arteries and larger diameters (+27.6%) in Lama4-/- arteries compared to wild type controls. Auto-fluorescence of the internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter. Scale bar is 100 µm. Data are means ± s.e.m from a minimum of 4 first order mesenteric arteries imaged at least in 4 mice per genotype. * P<0.05 unpaired t-test.


Figure 3A ) In vitro cell adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 and 511 , compared to the non - endothelial cell laminin 111 and fibronectin , showing high levels of HUAECs adhesion to laminin 511 and low binding to laminin 411 . B ) Enhanced fold change ( Δ ) in COX2 mRNA expression in HUAECs plated on laminin 511 versus laminin 111 in response to shear . mRNA data are means ± s . e . m of 4 independent experiments with triplicates / experiment , paired t - test . B ) Enhanced fold change ( Δ ) in COX2 protein levels in HUAECs plated on laminin 511 versus laminin 111 in response to shear . Protein quantification data are means ± s . e . m of 7 independent experiments , Mann - Whitney test . C ) Angle histogram of HUAEC orientation after 120 min of 10dyn / cm2 shear stress showing the percent cells at 10 - 90 ° in relation to the direction of flow . Cells were plated on 25 nM laminin 511 and laminin 111 . Data are means ± s . e . m of 6 independent experiments , t - test . D , E ) Inhibition assays performed at 25nM laminin 511 ( D ) in the presence or absence of function blocking integrin antibodies . Data are means ± s . e . m of 4 experiments with triplicates / experiment , unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . D , E ) Inhibition assays performed at 25nM laminin 111 , laminin 411 or fibronectin ( E ) in the presence or absence of function blocking integrin antibodies . Data are means ± s . e . m of 4 experiments with triplicates / experiment , unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

Figure 3A) In vitro cell adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 and 511, compared to the non-endothelial cell laminin 111 and fibronectin, showing high levels of HUAECs adhesion to laminin 511 and low binding to laminin 411.B) Enhanced fold change (Δ) in COX2 mRNA expression in HUAECs plated on laminin 511 versus laminin 111 in response to shear. mRNA data are means ± s.e.m of 4 independent experiments with triplicates/ experiment, paired t-test.B) Enhanced fold change (Δ) in COX2 protein levels in HUAECs plated on laminin 511 versus laminin 111 in response to shear. Protein quantification data are means ± s.e.m of 7 independent experiments, Mann-Whitney test.C) Angle histogram of HUAEC orientation after 120 min of 10dyn/cm2 shear stress showing the percent cells at 10-90° in relation to the direction of flow. Cells were plated on 25 nM laminin 511 and laminin 111. Data are means ± s.e.m of 6 independent experiments, t-test.D, E) Inhibition assays performed at 25nM laminin 511 (D) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.D, E) Inhibition assays performed at 25nM laminin 111, laminin 411 or fibronectin (E) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.


Figure 4 ( A ) Double immunofluorescence staining of whole mount wild type mesenteric resistance arteries for β1 integrin and vinculin show colocalization in focal adhesions ( arrow heads ) . Scale bar is 10 µm . B ) Vinculin immunofluorescence staining of whole mount wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek - Cre : : Lama5 - / - ( arrow heads ) and larger adhesion complexes in Lama4 - / - vessels ( arrow heads ) . Scale bar is 10 µm . Data are means ± s . e . m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice / genotype . * * * P < 0 . 001 , * * * * P < 0 . 0001 , unpaired t - test . B ) Vinculin immunofluorescence staining of whole mount wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek - Cre : : Lama5 - / - ( arrow heads ) and larger adhesion complexes in Lama4 - / - vessels ( arrow heads ) . Scale bar is 10 µm . Correpsonding quantification of sizes ( C ) and frequency distribution ( D ) of adhesion complexes per endothelial cell . Data are means ± s . e . m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice / genotype . * * * P < 0 . 001 , * * * * P < 0 . 0001 , unpaired t - test . E , F ) The endothelium of excised wild type , Tek - Cre : : Lama5 - / - aortae were analysed by AFM , revealing reduced cortical stiffness in Tek - Cre : : Lama5 - / - vessels ( - 9 . 5 % ) . Data are means ± s . e . m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment . * P < 0 . 05 , * * * * P < 0 . 0001 , unpaired t - test . E , F ) The endothelium of excised wild type and Lama4 - / - aortae were analysed by AFM , revealing increased cortical stiffness in Lama4 - / - vessels ( + 2 % ) . Data are means ± s . e . m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment . * P < 0 . 05 , * * * * P < 0 . 0001 , unpaired t - test . G ) In vitro AFM measurements of cortical stiffness performed on HUAECs plated on 30 nM purified laminin 511 or 111 , reveal increased cortical stiffness in cells on laminin 511 . Data are mean ± s . e . m from 3 experiments with triplicates / experiments , * P < 0 . 05 , unpaired t - test .

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

Figure 4(A) Double immunofluorescence staining of whole mount wild type mesenteric resistance arteries for β1 integrin and vinculin show colocalization in focal adhesions (arrow heads). Scale bar is 10 µm.B) Vinculin immunofluorescence staining of whole mount wild type, Tek-Cre::Lama5-/- and Lama4-/- mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek-Cre::Lama5-/- (arrow heads) and larger adhesion complexes in Lama4-/- vessels (arrow heads). Scale bar is 10 µm. Data are means ± s.e.m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice/genotype. ***P<0.001, ****P<0.0001, unpaired t-test.B) Vinculin immunofluorescence staining of whole mount wild type, Tek-Cre::Lama5-/- and Lama4-/- mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek-Cre::Lama5-/- (arrow heads) and larger adhesion complexes in Lama4-/- vessels (arrow heads). Scale bar is 10 µm. Correpsonding quantification of sizes (C) and frequency distribution (D) of adhesion complexes per endothelial cell. Data are means ± s.e.m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice/genotype. ***P<0.001, ****P<0.0001, unpaired t-test.E, F) The endothelium of excised wild type, Tek-Cre::Lama5-/- aortae were analysed by AFM, revealing reduced cortical stiffness in Tek-Cre::Lama5-/- vessels (-9.5%). Data are means ± s.e.m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment. *P<0.05, ****P<0.0001, unpaired t-test.E, F) The endothelium of excised wild type and Lama4-/- aortae were analysed by AFM, revealing increased cortical stiffness in Lama4-/- vessels (+2%). Data are means ± s.e.m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment. *P<0.05, ****P<0.0001, unpaired t-test.G) In vitro AFM measurements of cortical stiffness performed on HUAECs plated on 30 nM purified laminin 511 or 111, reveal increased cortical stiffness in cells on laminin 511. Data are mean ± s.e.m from 3 experiments with triplicates/experiments, *P<0.05, unpaired t-test.


Figure 5A ) A dual pipette - pulling assay was employed to measure cell - cell adhesion strength in HUAECs bound to laminin - coated beads ( asterisk ) or in the absence of beads . Scale bar is 10 µm . B ) Quantification shows higher adhesion strength between cells bound to laminin 511 - coated beads compared to laminin 111 - coated beads or cells not incubated with beads . Measurements could not be made with cells incubated with laminin 411 - coated beads since it was impossible to find cell - bead complexes . Values are means ± s . e . m from 15 cells from 3 independent experiments , unpaired t - test with Welch ' s correction . C ) Dual pipette - pulling assay performed in the presence of VE - cadherin blocking antibody , isotope control and soluble laminin 511 , showing almost complete ablation of adhesion strength in the presence of the blocking antibody in all conditions . Similar adhesion strengths were measured between cells in the presence of soluble laminin 511 and cells bound to laminin 511 - coated beads . D ) Adhesion strength in the presence of soluble laminin 511 was significantly reduced in the presence of integrin β1 blocking antibody ; cell - cell adhesion strength between cells bound to laminin 511 coated beads was significantly reduced in the presence of ROCK , SRC or FAK inhibitors . Values are means ± s . e . m from 11 cells from 3 independent experiments , unpaired t - test with Welch ' s correction . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , n . s . = not significant , n . d . = not determined .

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

Figure 5A) A dual pipette-pulling assay was employed to measure cell-cell adhesion strength in HUAECs bound to laminin-coated beads (asterisk) or in the absence of beads. Scale bar is 10 µm. B) Quantification shows higher adhesion strength between cells bound to laminin 511-coated beads compared to laminin 111-coated beads or cells not incubated with beads. Measurements could not be made with cells incubated with laminin 411-coated beads since it was impossible to find cell-bead complexes. Values are means ± s.e.m from 15 cells from 3 independent experiments, unpaired t-test with Welch's correction.C) Dual pipette-pulling assay performed in the presence of VE-cadherin blocking antibody, isotope control and soluble laminin 511, showing almost complete ablation of adhesion strength in the presence of the blocking antibody in all conditions. Similar adhesion strengths were measured between cells in the presence of soluble laminin 511 and cells bound to laminin 511-coated beads.D) Adhesion strength in the presence of soluble laminin 511 was significantly reduced in the presence of integrin β1 blocking antibody; cell-cell adhesion strength between cells bound to laminin 511 coated beads was significantly reduced in the presence of ROCK, SRC or FAK inhibitors. Values are means ± s.e.m from 11 cells from 3 independent experiments, unpaired t-test with Welch's correction. *P <0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s. = not significant, n.d. = not determined.


Figure 6A ) Immunofluorescence - based antibody feeding assay performed using HUAECs seeded on laminin 411 , 511 or 111 - coated coverslips , reveals more VE - cadherin at junctions and less in vesicles in cells plated on laminin 511 ( arrow heads mark VE - cadherin positive vesicles ) . Scale bar is 10 µm . B ) Quantification of VE - cadherin positive vesicles / cell in HUAECs plated on laminin 411 , 511 or 111 . Values are means ± s . e . m from 300 cells from 3 independent experiments , unpaired t - test . C ) Western blot of biotinylated ( surface ) VE - cadherin in HUAECs plated on different laminins versus total VE - cadherin ( input ) confirms more junctionally located VE - cadherin in cells bound to laminin 511 . D ) Quantification of Western blots expressed as relative signal proportions ( surface VE - cadherin / total VE - cadherin ) . Values shown are means ± s . e . m of 6 experiments , unpaired t - test . E ) Western blot of p120 catenincoimmunoprecipitated with VE - cadherin ( input ) from HUAECs plated on different laminins Asterisks are isotope controls . Values shown are means ± s . e . m of 4 experiments , Mann - Whitney test . E ) Western blot of p120 catenin coimmunoprecipitated with VE - cadherin ( input ) from HUAECs plated on different laminins , and ( F ) corresponding quantification of the p120 signal intensity relative to the total VE - cadherin signal . Asterisks are isotope controls . Values shown are means ± s . e . m of 4 experiments , Mann - Whitney test .

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

Figure 6A) Immunofluorescence-based antibody feeding assay performed using HUAECs seeded on laminin 411, 511 or 111-coated coverslips, reveals more VE-cadherin at junctions and less in vesicles in cells plated on laminin 511 (arrow heads mark VE-cadherin positive vesicles). Scale bar is 10 µm. B) Quantification of VE-cadherin positive vesicles/ cell in HUAECs plated on laminin 411, 511 or 111. Values are means ± s.e.m from 300 cells from 3 independent experiments, unpaired t-test.C) Western blot of biotinylated (surface) VE-cadherin in HUAECs plated on different laminins versus total VE-cadherin (input) confirms more junctionally located VE-cadherin in cells bound to laminin 511.D) Quantification of Western blots expressed as relative signal proportions (surface VE-cadherin/ total VE-cadherin). Values shown are means ± s.e.m of 6 experiments, unpaired t-test.E) Western blot of p120 catenincoimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test.E) Western blot of p120 catenin coimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins, and (F) corresponding quantification of the p120 signal intensity relative to the total VE-cadherin signal. Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test.


Figure 7A ) Double immunofluorescence staining for phosphorylated myosin light chain II ( pMLC II ) and VE - cadherin in mesenteric resistance artery endothelium from wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice ( arrow heads ) . Scale bar is 10µm . B ) Quantification of the pMLC II signal along VE - cadherin signal revealed significantly less overlap in Tek - Cre : : Lama5 - / - and more overlap in Lama4 - / - endothelium , compared with wild type controls . The data are normalized to the skeletal length of the VE - cadherin signal . Values shown are means ± s . e . m resulting from quantification of 4 field of view per arteries , 4 mice / genotype . * P < 0 . 05 , * * P < 0 . 01 , paired t - test .

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

Figure 7A) Double immunofluorescence staining for phosphorylated myosin light chain II (pMLC II) and VE-cadherin in mesenteric resistance artery endothelium from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice (arrow heads). Scale bar is 10µm. B) Quantification of the pMLC II signal along VE-cadherin signal revealed significantly less overlap in Tek-Cre::Lama5-/- and more overlap in Lama4-/- endothelium, compared with wild type controls. The data are normalized to the skeletal length of the VE-cadherin signal. Values shown are means ± s.e.m resulting from quantification of 4 field of view per arteries, 4 mice /genotype. *P<0.05, **P<0.01, paired t-test.


Figure 1A Purified TAP - tagged Rik1 - containing complexes analyzed by SDS - PAGE and silver staining . mock : a mock purification from an untagged strain . Proteins identified by LC - MS / MS are indicated at right , with the number of identified unique peptides in parentheses . B In vitro ubiquitylation assays using biotinylated ubiquitin , purified CLRC , and recombinant S . pombe histone H3 as the substrate . Proteins were analyzed by Western blotting ( WB ) using the indicated antibodies . Asterisks indicate ubiquitylated histone H3 species . C Ubiquitylation assays using recombinant histone H3 , the N - terminal tail of histone H3 ( residues 1 - 36 ) fused with GST ( H3N - GST ) , or GST alone as the substrate . Both fission yeast ( sp ) and human ( hs ) full - length H3 proteins and H3N - GST fusion proteins were examined . The proteins were analyzed by SDS - PAGE followed by either silver staining or Western blotting with an anti - biotin antibody . Asterisks indicate ubiquitylated histone H3 species .

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

Figure 1A Purified TAP-tagged Rik1-containing complexes analyzed by SDS-PAGE and silver staining. mock: a mock purification from an untagged strain. Proteins identified by LC-MS/MS are indicated at right, with the number of identified unique peptides in parentheses.B In vitro ubiquitylation assays using biotinylated ubiquitin, purified CLRC, and recombinant S. pombe histone H3 as the substrate. Proteins were analyzed by Western blotting (WB) using the indicated antibodies. Asterisks indicate ubiquitylated histone H3 species.C Ubiquitylation assays using recombinant histone H3, the N-terminal tail of histone H3 (residues 1-36) fused with GST (H3N-GST), or GST alone as the substrate. Both fission yeast (sp) and human (hs) full-length H3 proteins and H3N-GST fusion proteins were examined. The proteins were analyzed by SDS-PAGE followed by either silver staining or Western blotting with an anti-biotin antibody. Asterisks indicate ubiquitylated histone H3 species.


Figure 2A Ubiquitylation assay using H3N - GST . The components included in the assay and the ubiquitylated H3N - GST species are indicated at right . B MS / MS spectrum of the histone H3 peptide corresponding to residues 10 - 17 . The observed y and b ions and fragment map are shown . C Ubiquitylation assay using biotinylated ubiquitin and recombinant wild - type H3N - GST ( WT ) and arginine - substituted H3N - GST mutants as substrates . Proteins were analyzed by Western blotting and silver staining . mock : reaction without substrate . Asterisks indicate ubiquitylated H3N - GST proteins .

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

Figure 2A Ubiquitylation assay using H3N-GST. The components included in the assay and the ubiquitylated H3N-GST species are indicated at right.B MS/MS spectrum of the histone H3 peptide corresponding to residues 10-17. The observed y and b ions and fragment map are shown.C Ubiquitylation assay using biotinylated ubiquitin and recombinant wild-type H3N-GST (WT) and arginine-substituted H3N-GST mutants as substrates. Proteins were analyzed by Western blotting and silver staining. mock: reaction without substrate. Asterisks indicate ubiquitylated H3N-GST proteins.


Figure 3A Chromatin precipitated with anti - H3K4me2 or anti - H3K9me2 antibodies was analyzed by SDS - PAGE and silver staining . The input sample and mock precipitation using total mouse IgG are also shown . The gel slices indicated by boxes were excised and subjected to LC - MS / MS analysis . MS / MS spectrum of the histone H3 peptide corresponding to residues 9 - 17 . The observed y and b ions and fragment map are shown . C Summary of the ubiquitylated peptides identified by LC - MS / MS . D Relative abundance of ubiquitylated lysine residues in chromatin - associated histone H3 fractions . Chromatin precipitated with anti - H3K4me2 or anti - H3K9me2 antibodies was subjected to LC - MS / MS analysis , and the abundance of ubiquitylated lysine residues in each fraction is shown Since short peptides cleaved at unmodified lysine residues could not be efficiently detected in the LC - MS / MS analysis , these data do not accurately reflect the abundance including unmodified lysines .

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

Figure 3A Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was analyzed by SDS-PAGE and silver staining. The input sample and mock precipitation using total mouse IgG are also shown. The gel slices indicated by boxes were excised and subjected to LC-MS/MS analysis.MS/MS spectrum of the histone H3 peptide corresponding to residues 9-17. The observed y and b ions and fragment map are shown. C Summary of the ubiquitylated peptides identified by LC-MS/MS. D Relative abundance of ubiquitylated lysine residues in chromatin-associated histone H3 fractions. Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was subjected to LC-MS/MS analysis, and the abundance of ubiquitylated lysine residues in each fraction is shown Since short peptides cleaved at unmodified lysine residues could not be efficiently detected in the LC-MS/MS analysis, these data do not accurately reflect the abundance including unmodified lysines.


Figure 4B Heterochromatic silencing assays of wild - type and mutant Hht1 - FH strains . Silencing at the mating - type Kint2 : : ura4 + was evaluated . Ten - fold serial dilutions of the indicated strains were spotted onto non - selective medium ( N / S ) , medium lacking uracil ( - Ura ) , and medium containing 5 - FOA ( 5 - FOA ) . C ChIP analysis of H3K9me2 levels associated with the mating - type loci , relative to the control act1 + locus . Error bars indicate standard errors from three technical replicates . D ChIP analysis of Rik1 - myc levels associated with the mating - type cenH and centromeric dg loci , relative to the control act1 + locus . Error bars indicate standard errors from three technical replicates . E Heterochromatic silencing assays comparing the effects of H3 lysine substitution mutants . Silencing at the mating - type kint2 : : ura4 + was

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

Figure 4B Heterochromatic silencing assays of wild-type and mutant Hht1-FH strains. Silencing at the mating-type Kint2::ura4+ was evaluated. Ten-fold serial dilutions of the indicated strains were spotted onto non-selective medium (N/S), medium lacking uracil (-Ura), and medium containing 5-FOA (5-FOA).C ChIP analysis of H3K9me2 levels associated with the mating-type loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.D ChIP analysis of Rik1-myc levels associated with the mating-type cenH and centromeric dg loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.E Heterochromatic silencing assays comparing the effects of H3 lysine substitution mutants. Silencing at the mating-type kint2::ura4+ was evaluated