Figure 5A Quantitative real time PCR for enzymes related to lipid metabolism , using RNA from epididymalfat from 10 - week - old male WT and FADD - D mice on a SD . Data are expressed as mean ± SEM from two independent experiments ( n = 5 total for each genotype ) . * P = 0 . 0162 , * * P = 0 . 0105 , * * * P = 0 . 0063 ( Student ' s t - test ) . NS , not statistically significant . B cAMP abundance in epididymalWAT from 10 - week - old male WT and FADD - D mice on a HFD ( n = 4 for each genotype ) . Data are expressed as mean ± SEM . * P = 0 . 0033 ( Student ' s t - test ) . C Basal and forskolin - stimulated adenylyl cyclase activity in epididymalWAT from 10 - week - old male WT and FADD - D mice on a HFD ( n = 4 for each genotype ) . Data are expressed as mean ± SEM . * P = 0 . 0177 , * P = 0 . 0091 ( Student ' s t - test ) . D Immunoblotting of phosphorylated HSL ( p - HSL ) , HSL , p - PKA substrate , desnutrin and Tubulin ( control ) ( left ) with relative quantification ( right ) . Data are expressed as mean ± SEM from three independent experiments . * P = 0 . 0032 , * * P = 0 . 0011 ( Student ' s t - test ) . NS , not statistically significant . E Basal and stimulated ( + 200 nM isoproterenol ) lipolysis in epididymalWAT of 10 - week - old male WT and FADD - D mice ( n = 4 for each genotype ) treated with or without 10 μM BAY . Data are expressed as mean ± SEM . * P = 0 . 0019 , * * P = 00038 , ^ P = 0 . 0035 , ^ ^ P = 0 . 0053 ( one - way ANOVA ) . NS , not statistically significant . F Basal and stimulated ( + 200 nM isoproterenol ) lipolysis in isolated adipocytes from 10 - week - old WT and FADD - D mice ( n = 4 for each genotype ) treated with or without 10 μM BAY . Data are expressed as mean ± SEM . * P = 0 . 0033 , * * P = 0 . 0051 , ^ P = 0 . 004 , ^ ^ P = 0 . 0057 ( one - way ANOVA ) . NS , not statistically significant . G Immunoblotting of phosphorylated PKC ( p - PKC ) , ERK , phosphorylated ERK ( p - ERK ) and β - actin ( control ) ( left ) with relative quantification ( right ) . Data are expressed as mean ± SEM from three independent experiments . * P = 0 . 0119 , * * P = 0 . 0034 ( Student ' s 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 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 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Quantitative real time PCR for enzymes related to lipid metabolism, using RNA from epididymalfat from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from two independent experiments (n = 5 total for each genotype). *P = 0.0162, **P = 0.0105, ***P = 0.0063 (Student's t-test). NS, not statistically significant.B cAMP abundance in epididymalWAT from 10-week-old male WT and FADD-D mice on a HFD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0033 (Student's t-test).C Basal and forskolin-stimulated adenylyl cyclase activity in epididymalWAT from 10-week-old male WT and FADD-D mice on a HFD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0177, *P = 0.0091 (Student's t-test).D Immunoblotting of phosphorylated HSL (p-HSL), HSL, p-PKA substrate, desnutrin and Tubulin (control) (left) with relative quantification (right). Data are expressed as mean ± SEM from three independent experiments. *P = 0.0032, **P = 0.0011 (Student's t-test). NS, not statistically significant.E Basal and stimulated (+200 nM isoproterenol) lipolysis in epididymalWAT of 10-week-old male WT and FADD-D mice (n = 4 for each genotype) treated with or without 10 μM BAY. Data are expressed as mean ± SEM. *P = 0.0019, **P = 00038, ^P = 0.0035, ^^P = 0.0053 (one-way ANOVA). NS, not statistically significant.F Basal and stimulated (+200 nM isoproterenol) lipolysis in isolated adipocytes from 10-week-old WT and FADD-D mice (n = 4 for each genotype) treated with or without 10 μM BAY. Data are expressed as mean ± SEM. *P = 0.0033, **P = 0.0051, ^P = 0.004, ^^P = 0.0057 (one-way ANOVA). NS, not statistically significant.G Immunoblotting of phosphorylated PKC (p-PKC), ERK, phosphorylated ERK (p-ERK) and β-actin (control) (left) with relative quantification (right). Data are expressed as mean ± SEM from three independent experiments. *P = 0.0119, **P = 0.0034 (Student's t-test).


Figure 6A Typical growth curves of control and ad - FADDmice maintained on a SD ( left panel ) or a HFD ( right panel ) ( n = 8 for each genotype ) . Data are expressed as mean ± SEM . * P = 0 . 0435 , * * P = 0 . 0371 , ^ P = 0 . 0217 , # P = 0 . 0236 , # # P = 0 . 0158 , # # # P = 0 . 0113 ( Student ' s t - test ) . B Body composition by NMR to show the fat mass and fat mass ratio in control and ad - FADDmice after 10 weeks of HFD ( n = 6 for each genotype ) . Data are expressed as mean ± SEM . * P = 0 . 0027 , * * P = 0 . 0012 ( Student ' s t - test ) . C Food intake in 15 - week - old male control and ad - FADDmice fed a HFD ( n = 8 for each genotype ) . D and E Representative pictures of hematoxylin and eosin - stained sections of epididymalWAT from control and ad - FADDmice at 10 weeks ( D ) and after 10 weeks of an HFD ( E ) are shown . Scale bar = 50 μm . Shown are typical results from four different fields and three different experiments . F The average cross - sectional area of adipocytes in epididymalWAT is shown in the bar graph ( n = 6 for each genotype ) . Data are expressed as mean ± SEM . * P = 0 . 002 ( Student ' s t - test ) . NS , not statistically significant . G The distribution of adipocyte cross - sectional area in epididymalWAT of HFD - fed control or ad - FADDmice is shown in the bar graph ( n = 6 for each genotype ) . H Representative pictures of hematoxylin and eosin - stained sections of brown adipose tissue from HFD - fed control and ad - FADDmice are shown . Scale bar = 50 μm . Shown are typical results from four different fields and three different experiments .

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

Figure 6A Typical growth curves of control and ad-FADDmice maintained on a SD (left panel) or a HFD (right panel) (n = 8 for each genotype). Data are expressed as mean ± SEM. *P = 0.0435, **P =0.0371, ^P = 0.0217, #P = 0.0236, ##P = 0.0158, ###P = 0.0113 (Student's t-test).B Body composition by NMR to show the fat mass and fat mass ratio in control and ad-FADDmice after 10 weeks of HFD (n = 6 for each genotype). Data are expressed as mean ± SEM. *P = 0.0027, **P = 0.0012 (Student's t-test).C Food intake in 15-week-old male control and ad-FADDmice fed a HFD (n = 8 for each genotype).D and E Representative pictures of hematoxylin and eosin-stained sections of epididymalWAT from control and ad-FADDmice at 10 weeks (D) and after 10 weeks of an HFD (E) are shown. Scale bar = 50 μm. Shown are typical results from four different fields and three different experiments.F The average cross-sectional area of adipocytes in epididymalWAT is shown in the bar graph (n = 6 for each genotype). Data are expressed as mean ± SEM. *P = 0.002 (Student's t-test). NS, not statistically significant.G The distribution of adipocyte cross-sectional area in epididymalWAT of HFD-fed control or ad-FADDmice is shown in the bar graph (n = 6 for each genotype).H Representative pictures of hematoxylin and eosin-stained sections of brown adipose tissue from HFD-fed control and ad-FADDmice are shown. Scale bar = 50 μm. Shown are typical results from four different fields and three different experiments.


Figure 7A - C HFD - fed control and ad - FADDmice were housed in a computer - controlled open - circuit indirect calorimeter to determine ( A ) oxygen consumption , ( B ) carbon dioxide production , and ( C ) respiratory exchange ratio ( RER ) during the light ( 8 am - 8 pm ) and dark ( 8 pm - 8 am ) periods ( n = 5 for each genotype ) . Data are expressed as mean ± SEM . * P = 0 . 0379 , * * P = 0 . 0453 , * * * P = 0 . 0445 ( Student ' s t - test ) . NS , not statistically significant . D The result of real - time quantitative PCR analysis for UCP1 in brown adipose tissue from HFD - fed control and ad - FADDmice is shown in the bar graph . Data are expressed as mean ± SEM from two independent experiments ( n = 4 total for each genotype ) . * P = 0 . 0172 ( Student ' s t - test ) . E Immunoblot of UCP1 and Tubulin ( control ) ( top ) in BAT from control and ad - FADDmice with relative quantification ( bottom ) . Data are expressed as mean ± SEM from three independent experiments . * P = 0 . 0053 ( Student ' s t - test ) . F Mice were initially maintained at room temperature and then transferred to cold incubator ( 5 ° C ) . Rectal body temperature was recorded at the indicated time points using thermoprobe ( n = 6 for each genotype ) . Results are means ± SEM . * P = 0 . 0466 , * * P = 0 . 0357 , # P = 0 . 0229 , # # P = 0 . 0138 , # # # P = 0 . 0206 ( Student ' s 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 7A-C HFD-fed control and ad-FADDmice were housed in a computer-controlled open-circuit indirect calorimeter to determine (A) oxygen consumption, (B) carbon dioxide production, and (C) respiratory exchange ratio (RER) during the light (8 am - 8 pm) and dark (8 pm - 8 am) periods (n = 5 for each genotype). Data are expressed as mean ± SEM. *P = 0.0379, **P = 0.0453, ***P = 0.0445 (Student's t-test). NS, not statistically significant.D The result of real-time quantitative PCR analysis for UCP1 in brown adipose tissue from HFD-fed control and ad-FADDmice is shown in the bar graph. Data are expressed as mean ± SEM from two independent experiments (n = 4 total for each genotype). *P = 0.0172 (Student's t-test).E Immunoblot of UCP1 and Tubulin (control) (top) in BAT from control and ad-FADDmice with relative quantification (bottom). Data are expressed as mean ± SEM from three independent experiments. *P = 0.0053 (Student's t-test).F Mice were initially maintained at room temperature and then transferred to cold incubator (5°C). Rectal body temperature was recorded at the indicated time points using thermoprobe (n = 6 for each genotype). Results are means ± SEM. *P = 0.0466, **P = 0.0357, #P = 0.0229, ##P = 0.0138, ###P = 0.0206 (Student's t-test).


Figure 8A and B Blood glucose levels and serum insulin levels in 2 - h GTT 12 weeks after HFD . Inset graphs in ( A ) and ( B ) depict the respective analysis of the area under the curve ( AUC ) . Data shown are from one experiment ( n = 6 for each genotype ) , representative of a total of two independent experiments . Results are means ± SEM . * P = 0 . 0287 , * * P = 0 . 0173 , * * * P = 0 . 0332 , * * * * P = 0 . 0453 , # P = 0 . 0077 , # # P = 0 . 0344 , # # # P = 0 . 0412 , # # # # P = 0 . 0495 , ^ P = 0 . 0435 , ^ ^ P = 0 . 0304 ( Student ' s t - test ) . C ITT 12 weeks after HFD . Data shown are from one experiment ( n = 6 for each genotype ) , representative of a total of two independent experiments . Results are means ± SEM . * P = 0 . 0127 , * * P = 0 . 0232 , * * * P = 0 . 0373 , * * * * P = 0 . 0385 ( Student ' s t - test ) . D Insulin - stimulated Akt phosphorylation ( Ser473 ) in WAT , liver , and skeletal muscle of control and ad - FADD mice . The experiments were performed in triplicate . E Adiponectin mRNA was measured in isolated epididymal adipocytes by RT - PCR . Data are expressed as mean ± SEM from three independent experiments . * P = 0 . 0044 ( Student ' s t - test ) . F - H Total adiponectin levels ( F ) , HMW adiponectin ( G ) , and the ratio of HMW to total adiponectin ( H ) . n = 5 for each genotype . Data are expressed as mean ± SEM . * P = 0 . 0217 , * * P = 0 . 0116 , * * * P = 0 . 0175 ( Student ' s t - test ) . F - H Total adiponectin levels ( F ) , HMW adiponectin ( G ) , and the ratio of HMW to total adiponectin ( H ) . n = 5 for each genotype . Data are expressed as mean ± SEM . * P = 0 . 0217 , * * P = 0 . 0116 , * * * P = 0 . 0175 ( Student ' s t - test ) . I Basal and insulin - stimulated glucose uptake in control and FADD deficient primary isolated adipocytes . Data shown are from one experiment ( n = 4 for each genotype ) , representative of a total of two independent experiments . Results are means ± SEM . * P = 0 . 0073 ( Student ' s t - test ) . J Basal and insulin - stimulated glucose uptake in 3T3L1 cells differentiated to adipocytes and infected with lentivirus containing FADD shRNA or scrambled shRNA control . Data are expressed as mean ± SEM from three independent experiments . * P = 0 . 0092 ( Student ' s t - test ) . K Basal and insulin - stimulated glucose uptake in control and FADD deficient primary isolated adipocytes treated with DMSO or 5 μM MK886 . Data are expressed as mean ± SEM from four independent experiments . * P = 0 . 0135 ( one - way ANOVA ) . NS , not statistically significant .

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 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 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 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 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 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 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 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

Figure 8A and B Blood glucose levels and serum insulin levels in 2-h GTT 12 weeks after HFD. Inset graphs in (A) and (B) depict the respective analysis of the area under the curve (AUC). Data shown are from one experiment (n = 6 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0287, **P = 0.0173, ***P = 0.0332, ****P = 0.0453, #P = 0.0077, ##P = 0.0344, ###P = 0.0412, ####P = 0.0495, ^P = 0.0435, ^^P = 0.0304 (Student's t-test).C ITT 12 weeks after HFD. Data shown are from one experiment (n = 6 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0127, **P = 0.0232, ***P = 0.0373, ****P = 0.0385 (Student's t-test).D Insulin-stimulated Akt phosphorylation (Ser473) in WAT, liver, and skeletal muscle of control and ad-FADD mice. The experiments were performed in triplicate.E Adiponectin mRNA was measured in isolated epididymal adipocytes by RT-PCR. Data are expressed as mean ± SEM from three independent experiments. *P = 0.0044 (Student's t-test).F-H Total adiponectin levels (F), HMW adiponectin (G), and the ratio of HMW to total adiponectin (H). n = 5 for each genotype. Data are expressed as mean ± SEM. *P = 0.0217, **P = 0.0116, ***P = 0.0175 (Student's t-test).F-H Total adiponectin levels (F), HMW adiponectin (G), and the ratio of HMW to total adiponectin (H). n = 5 for each genotype. Data are expressed as mean ± SEM. *P = 0.0217, **P = 0.0116, ***P = 0.0175 (Student's t-test).I Basal and insulin-stimulated glucose uptake in control and FADD deficient primary isolated adipocytes. Data shown are from one experiment (n = 4 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0073 (Student's t-test).J Basal and insulin-stimulated glucose uptake in 3T3L1 cells differentiated to adipocytes and infected with lentivirus containing FADD shRNA or scrambled shRNA control. Data are expressed as mean ± SEM from three independent experiments. *P = 0.0092 (Student's t-test).K Basal and insulin-stimulated glucose uptake in control and FADD deficient primary isolated adipocytes treated with DMSO or 5 μM MK886. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0135 (one-way ANOVA). NS, not statistically significant.


Figure 9A Relative mRNA levels of the macrophage marker F4 / 80 in SVCs isolated from epididymal adipose tissue . Data are expressed as mean ± SEM from four independent experiments . * P = 0 . 0279 ( Student ' s t - test ) . B F4 / 80 immunostaining in epi - WAT . Scale bars = 50 μm . Shown are typical results from four different fields and three different experiments . Data are expressed as mean ± SEM . * P = 0 . 0003 ( Student ' s t - test ) . C Relative mRNA levels of the proinflammatory M1 - like macrophage marker CD11c in SVCs isolated from epididymal adipose tissue . Data are expressed as mean ± SEM from five independent experiments . * P = 0 . 0221 ( Student ' s t - test ) . D Relative mRNA levels of inflammatory and anti - inflammatory cytokines in Epi - WAT . Data are expressed as mean ± SEM from four independent experiments . * P = 0 . 0201 , * * P = 0 . 0159 , * * * P = 0 . 0263 , ^ P = 0 . 0227 , ^ ^ P = 0 . 0215 , ^ ^ ^ P = 0 . 0312 , # P = 0 . 0231 , # # P = 0 . 0255 , # # # P = 0 . 0094 ( Student ' s t - test ) . NS , not statistically significant . E - H Serum levels of TNF - α ( E ) . n = 5 for each genotype . Data are expressed as mean ± SEM . * P = 0 . 0077 , * * P = 0 . 0052 , * * * P = 0 . 0114 , * * * * P = 0 . 0127 ( Student ' s t - test ) . E - H Serum levels of IL - 6 ( F ) , IL - 12p40 ( G ) , and IL - 10 ( H ) . n = 5 for each genotype . Data are expressed as mean ± SEM . * P = 0 . 0077 , * * P = 0 . 0052 , * * * P = 0 . 0114 , * * * * P = 0 . 0127 ( Student ' s 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 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 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 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

Figure 9A Relative mRNA levels of the macrophage marker F4/80 in SVCs isolated from epididymal adipose tissue. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0279 (Student's t-test).B F4/80 immunostaining in epi-WAT. Scale bars = 50 μm. Shown are typical results from four different fields and three different experiments. Data are expressed as mean ± SEM. *P = 0.0003 (Student's t-test).C Relative mRNA levels of the proinflammatory M1-like macrophage marker CD11c in SVCs isolated from epididymal adipose tissue. Data are expressed as mean ± SEM from five independent experiments. *P = 0.0221 (Student's t-test).D Relative mRNA levels of inflammatory and anti-inflammatory cytokines in Epi-WAT. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0201, **P = 0.0159, ***P = 0.0263, ^P = 0.0227, ^^P = 0.0215, ^^^P = 0.0312, #P = 0.0231, ##P = 0.0255, ###P = 0.0094 (Student's t-test). NS, not statistically significant.E-H Serum levels of TNF-α (E). n = 5 for each genotype. Data are expressed as mean ± SEM. *P = 0.0077, **P = 0.0052, ***P = 0.0114, ****P = 0.0127 (Student's t-test).E-H Serum levels of IL-6 (F), IL-12p40 (G), and IL-10 (H). n = 5 for each genotype. Data are expressed as mean ± SEM. *P = 0.0077, **P = 0.0052, ***P = 0.0114, ****P = 0.0127 (Student's t-test).


Figure 10A Top , representative photographs of 20 - week - old male ob / ob and FADD - D / ob / ob mice fed a SD . Bottom , representative photographs of their livers and epididymalWAT . B Comparison of weights of WAT depots from WT , FADD - D , ob / ob and FADD - D / ob / ob mice . n = 5 for each genotype . Data are expressed as mean ± SEM . * P = 0 . 0007 , * * P = 0 . 0022 , * * * P = 0 . 0011 , # P = 0 . 0009 , # # P = 0 . 0003 , # # # P = 0 . 0027 ( one - way ANOVA ) . C Basal and stimulated lipolysis measured by fatty acid release from explants of epididymalWAT in 10 - week - old male ob / ob and FADD - D / ob / ob mice fed a HFD . n = 4 for each genotype . Data are expressed as mean ± SEM . * P = 0 . 0003 , * * P = 0 . 001 ( one - way ANOVA ) . D cAMP level in WAT of 10 - week - old male WT , FADD - D , ob / ob and FADD - D / ob / ob mice fed a HFD . n = 4 for each genotype . Data are expressed as mean ± SEM . * P = 0 . 0017 , * * P = 0 . 001 ( one - way ANOVA ) . E Whole - body oxygen consumption rate ( VO2 ) during 24 hr in WT , FADD - D , ob / ob , and FADD - D / ob / ob mice fed a SD . n = 4 for each genotype . Data are expressed as mean ± SEM . * P = 0 . 0053 , * * P = 0 . 0025 , * * * P = 0 . 0062 , * * * * P = 0 . 0011 ( one - way ANOVA ) . F Quantitative real time PCR for Dio2 , PPARd and UCP1 , using RNA from epididymal fat from 20 - week - old male ob / ob and FADD - D / ob / ob mice fed a SD . Data are expressed as mean ± SEM from four independent experiments . * P = 0 . 0009 , * * P = 0 . 0015 , * * * P = 0 . 0003 ( Student ' s t - test ) . G Quantitative real time PCR for UCP3 , PGC - 1α , PPARd and CPT1 , using RNA from skeletal muscle from 20 - week - old male ob / ob and FADD - D / ob / ob mice fed a SD . Data are expressed as mean ± SEM from four independent experiments . * P = 0 . 0105 , * * P = 0 . 0033 , * * * P = 0 . 0058 ( Student ' s t - test ) . NS , not statistically significant . H Quantitative real time PCR for UCP1 , PPARa , CPT1 and PGC - 1α , using RNA from brown adipose fat from 20 - week - old male ob / ob and FADD - D / ob / ob mice fed a SD . Data are expressed as mean ± SEM from four independent experiments . * P = 0 . 0012 * * P = 0 . 0129 , * * * P = 0 . 0103 , * * * * P = 0 . 0207 ( Student ' s t - test ) . I Oxidation of [ 1 - 14C ] oleic acid to 14CO2by adipocytes isolated from WT , FADD - D , ob / ob , and FADD - D / ob / ob mice . Data shown are from one experiment ( n = 3 for each genotype ) , representative of a total of two independent experiments . Results are means ± SEM . * P = 0 . 0031 , * * P = 0 . 0075 ( one - way ANOVA ) . NS , not statistically significant . J Immunoblot of VLCAD , LCAD , MCAD , PTL and Tubulin ( control ) , using protein extracts from epididymal fat from 15 - week - old male WT , FADD - D , ob / ob and FADD - D / ob / ob mice fed a SD . Data shown are representative of three independent experiments having similar results .

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

Figure 10A Top, representative photographs of 20-week-old male ob/ob and FADD-D/ob/ob mice fed a SD. Bottom, representative photographs of their livers and epididymalWAT.B Comparison of weights of WAT depots from WT, FADD-D, ob/ob and FADD-D/ob/ob mice. n = 5 for each genotype. Data are expressed as mean ± SEM. *P = 0.0007, **P = 0.0022, ***P = 0.0011, #P = 0.0009, ##P = 0.0003, ###P = 0.0027 (one-way ANOVA).C Basal and stimulated lipolysis measured by fatty acid release from explants of epididymalWAT in 10-week-old male ob/ob and FADD-D/ob/ob mice fed a HFD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0003, **P = 0.001 (one-way ANOVA).D cAMP level in WAT of 10-week-old male WT, FADD-D, ob/ob and FADD-D/ob/ob mice fed a HFD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0017, **P = 0.001 (one-way ANOVA).E Whole-body oxygen consumption rate (VO2) during 24 hr in WT, FADD-D, ob/ob, and FADD-D/ob/ob mice fed a SD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0053, **P = 0.0025, ***P = 0.0062, ****P = 0.0011 (one-way ANOVA).F Quantitative real time PCR for Dio2, PPARd and UCP1, using RNA from epididymal fat from 20-week-old male ob/ob and FADD-D/ob/ob mice fed a SD. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0009, **P = 0.0015, ***P = 0.0003 (Student's t-test).G Quantitative real time PCR for UCP3, PGC-1α, PPARd and CPT1, using RNA from skeletal muscle from 20-week-old male ob/ob and FADD-D/ob/ob mice fed a SD. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0105, **P = 0.0033, ***P = 0.0058 (Student's t-test). NS, not statistically significant.H Quantitative real time PCR for UCP1, PPARa, CPT1 and PGC-1α, using RNA from brown adipose fat from 20-week-old male ob/ob and FADD-D/ob/ob mice fed a SD. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0012 **P = 0.0129, ***P = 0.0103, ****P = 0.0207 (Student's t-test).I Oxidation of [1-14C] oleic acid to 14CO2by adipocytes isolated from WT, FADD-D, ob/ob, and FADD-D/ob/ob mice. Data shown are from one experiment (n = 3 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0031, **P = 0.0075 (one-way ANOVA). NS, not statistically significant.J Immunoblot of VLCAD, LCAD, MCAD, PTL and Tubulin (control), using protein extracts from epididymal fat from 15-week-old male WT, FADD-D, ob/ob and FADD-D/ob/ob mice fed a SD. Data shown are representative of three independent experiments having similar results.


Figure 1Confocal microscopy of IBA - 1 ( green staining ) immunohistochemistry of RPE flatmounts ( RPE autofluorescence visible as orange due to its autofluorescence in the red and green channel ) from a healthy donor ( A ) , a geographic atrophy lesion ( B ) , and large drusen ( C and D ) . ( A , B , D ) : orthogonal Z - stack projection ; ( C ) : oblique Z - stack projection and dissecting microscope appearance of postmortem large drusen after removal of the overlaying retina ( inset ) . Double - labeling on the subretinal side of the retina ( to avoid masking by RPE autofluorescence ) of IBA - 1 + ( E , green fluorescence ) and CD18 ( F , red fluorescence ; G , merge ) . Orthogonal and lateral Z - stack of a subretinal IBA - 1 + ( green fluorescence ) MPs adjacent to the RPE ( orange autofluorescence ) in the vicinity of a large drusen .

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 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

Figure 1Confocal microscopy of IBA-1 (green staining) immunohistochemistry of RPE flatmounts (RPE autofluorescence visible as orange due to its autofluorescence in the red and green channel) from a healthy donor (A), a geographic atrophy lesion (B), and large drusen (C and D). (A, B, D): orthogonal Z-stack projection; (C): oblique Z-stack projection and dissecting microscope appearance of postmortem large drusen after removal of the overlaying retina (inset).Double-labeling on the subretinal side of the retina (to avoid masking by RPE autofluorescence) of IBA-1+ (E, green fluorescence) and CD18 (F, red fluorescence; G, merge).Orthogonal and lateral Z-stack of a subretinal IBA-1+ (green fluorescence) MPs adjacent to the RPE (orange autofluorescence) in the vicinity of a large drusen.


Figure 2APOE ( A ) and IBA - 1 ( B , merge in C ) double - labeling on paraffin sections of surgical specimens of human tonsils . APOE ( D ) and IBA - 1 ( E , merge in F ) double - labeling on the subretinal aspect of a large drusen overlaying retina ( to avoid specific signal masking by the RPE orange autofluorescence ) . APOE ( red staining ) immunohistochemistry of control donor tissue ( G ) and adjacent to GA lesion ( H ) . IBA - 1 ( green staining , RPE far - red autofluorescence ) double - labeling of the GA lesion shown in ( H ) . Representative images from five donor eyes with large drusen , five donor eyes with GA , and three healthy donors between 70 and 89 years old .

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

Figure 2APOE (A) and IBA-1 (B, merge in C) double-labeling on paraffin sections of surgical specimens of human tonsils.APOE (D) and IBA-1 (E, merge in F) double-labeling on the subretinal aspect of a large drusen overlaying retina (to avoid specific signal masking by the RPE orange autofluorescence).APOE (red staining) immunohistochemistry of control donor tissue (G) and adjacent to GA lesion (H).IBA-1 (green staining, RPE far-red autofluorescence) double-labeling of the GA lesion shown in (H). Representative images from five donor eyes with large drusen, five donor eyes with GA, and three healthy donors between 70 and 89 years old.


Figure 3Immunohistochemistry of APOE ( red ) and IBA - 1 ( green ) on a section ( A , blue Hoechst ) and the subretinal side of a retinal flatmount ( B and C ) from a 12 - month - old Cx3cr1GFP / GFP mouse ( representative of 3 independent experiments , experiments omitting the primary antibody immunostaining served as negative controls ) . ONL : outer nuclear layer ; RPE : retinal pigment epithelium . Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP monocytes cultured for 24 h in contact with POS of an overlaying retinal explant ( n = 4 / group per experiment ; Mann - Whitney U - test , * P = 0 . 0286 , the experiment was repeated twice with similar results ) . Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP FACS - sorted microglial cells , freshly extracted from adult brain ( n = 4 - 5 / group ; Mann - Whitney U - test , C57BL / 6J versus Cx3cr1GFP / GFP * P = 0 . 0159 ) . Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP peritoneal macrophages cultured for 24 h with CX3CL1 . ( n = 4 / group per experiment ; Mann - Whitney U - test , * P = 0 . 0286 . The experiment was repeated twice with similar results ) . APOE Western blot analysis of equivalent amounts of supernatant protein from CX3CL1 - exposed C57BL / 6J and Cx3cr1GFP / GFP peritoneal macrophages at 24 h . Soluble Mer receptor tyrosine kinase that is released constitutively from cultured macrophages served as a loading control . The experiment was repeated twice with similar results . 12 - month - old IBA - 1 stained RPE flatmounts of Cx3cr1GFP / GFP ( H ) and Cx3cr1GFP / GFPApoE − / − ( I ) mice . Quantification of subretinal IBA - 1 + mononuclear phagocytes in 2 - month - old ( left ) and 12 - month - old ( right ) mice of the indicated strains ( n = 9 - 20 / group ANOVA / Dunnett test at 12 months : Cx3cr1GFP / GFP versus any other group * P < 0 . 0001 ; Mann - Whitney U - test at 12 months of Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P < 0 . 0001 ) . Quantification of subretinal IBA - 1 + mononuclear phagocytes after 4 days of light challenge of 2 - month - old mice of the indicated strains ( n = 10 - 25 / group ANOVA / Dunnett test : Cx3cr1GFP / GFP versus any other group * P < 0 . 0001 ; Mann - Whitney U - test of Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P < 0 . 0001 ) . IBA - 1 - ( green ) and CD102 - ( red ) stained RPE flatmounts 7 days after laser injury of 2 - month - old Cx3cr1GFP / GFP ( E ) and Cx3cr1GFP / GFPApoE − / − ( F ) mice . Quantification of subretinal IBA - 1 + mononuclear phagocytes on the RPE counted on the RPE at a distance of 0 - 500 μm to CD102 + CNV 7 days after the laser injury of 2 - month - old mice of the indicated strains ( n = 8 - 10 eyes / group ANOVA / Dunnett test : Cx3cr1GFP / GFP versus any other group * P < 0 . 0001 ; Mann - Whitney U - test of Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Immunohistochemistry of APOE (red) and IBA-1 (green) on a section (A, blue Hoechst) and the subretinal side of a retinal flatmount (B and C) from a 12-month-old Cx3cr1GFP/GFP mouse (representative of 3 independent experiments, experiments omitting the primary antibody immunostaining served as negative controls). ONL: outer nuclear layer; RPE: retinal pigment epithelium.Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP monocytes cultured for 24 h in contact with POS of an overlaying retinal explant (n = 4/group per experiment; Mann-Whitney U-test, *P = 0.0286, the experiment was repeated twice with similar results).Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP FACS-sorted microglial cells, freshly extracted from adult brain (n = 4-5/group; Mann-Whitney U-test, C57BL/6J versus Cx3cr1GFP/GFP *P = 0.0159).Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP peritoneal macrophages cultured for 24 h with CX3CL1. (n = 4/group per experiment; Mann-Whitney U-test, *P = 0.0286. The experiment was repeated twice with similar results).APOE Western blot analysis of equivalent amounts of supernatant protein from CX3CL1-exposed C57BL/6J and Cx3cr1GFP/GFP peritoneal macrophages at 24 h. Soluble Mer receptor tyrosine kinase that is released constitutively from cultured macrophages served as a loading control. The experiment was repeated twice with similar results.12-month-old IBA-1 stained RPE flatmounts of Cx3cr1GFP/GFP (H) and Cx3cr1GFP/GFPApoE−/− (I) mice.Quantification of subretinal IBA-1+ mononuclear phagocytes in 2-month-old (left) and 12-month-old (right) mice of the indicated strains (n = 9-20/group ANOVA/Dunnett test at 12 months: Cx3cr1GFP/GFP versus any other group *P < 0.0001; Mann-Whitney U-test at 12 months of Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P < 0.0001).Quantification of subretinal IBA-1+ mononuclear phagocytes after 4 days of light challenge of 2-month-old mice of the indicated strains (n = 10-25/group ANOVA/Dunnett test: Cx3cr1GFP/GFP versus any other group *P < 0.0001; Mann-Whitney U-test of Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P < 0.0001).IBA-1- (green) and CD102- (red) stained RPE flatmounts 7 days after laser injury of 2-month-old Cx3cr1GFP/GFP (E) and Cx3cr1GFP/GFPApoE−/− (F) mice.Quantification of subretinal IBA-1+mononuclear phagocytes on the RPE counted on the RPE at a distance of 0-500 μm to CD102+CNV 7 days after the laser injury of 2-month-old mice of the indicated strains (n = 8-10 eyes/group ANOVA/Dunnett test: Cx3cr1GFP/GFP versus any other group *P < 0.0001; Mann-Whitney U-test of Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P < 0.0001).


Figure 4Representative image of a RPE flatmount 12 h after the subretinal injection ( red marking ) of 4 μl PBS with 12 , 000 CFSE - stained thioglycollate - elicited peritoneal cells that contain 70 % macrophages ( inset close - up view ) . Quantifications of CFSE + F4 / 80 + macrophages at different time points after subretinal injections of C57BL / 6J and Cx3cr1GFP / GFP CFSE + macrophages ( n = 5 / per group ( 12 h ) and n = 6 / per group thereafter ; Mann - Whitney U - test , C57BL / 6J versus Cx3cr1GFP / GFP : 1 day n = 20 / group * P < 0 . 0001 ; 2 day n = 6 / group * P = 0 . 0317 ) . Representative image of TUNEL / Hoechst double - staining 12 h after subretinal injection of C57BL / 6J CFSE + macrophages ( experiment repeated three times , inset close - up view ) . Representative cytometry images of SSC - A / CFSE and CD11b / F4 / 80 gated analysis of eye cell suspensions prepared 24 h after the injection of Cx3cr1GFP / GFP CFSE + macrophages and cytometric quantification of eye cell suspensions at 24 h after the injection of C57BL / 6J and Cx3cr1GFP / GFP macrophages into C57BL / 6J ( n = 16 - 20 / group ; Mann - Whitney U - test , * P = 0 . 0024 ) . Quantification of subretinal CFSE + cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE + magnetic - bead - sorted bone marrow - derived monocytes ( Mo ) from C57BL / 6J and Cx3cr1GFP / GFPmice into C57BL / 6Jmice ( n = 8 - 12 / group ; Mann - Whitney U - test , * P = 0 . 0006 ) . Quantification of subretinal CFSE + cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE + CD11bFACS - sorted brainmicroglial cells from C57BL / 6J and Cx3cr1GFP / GFPmice into C57BL / 6Jmice ( n = 9 - 12 / group ; Mann - Whitney U - test , * P = 0 . 0087 ) . Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injections of CFSE + macrophages from C57BL / 6J , Cx3cr1GFP / GFP , Cx3cr1GFP / GFPApoE − / − , and ApoE − / − mice into C57BL / 6Jmice ( n = 8 - 12 / group ; one - way ANOVA / Dunnett test of Cx3cr1GFP / GFP versus any other group * P ≤ 0 . 0001 ; Mann - Whitney U - test , Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P = 0 . 0006 ) . Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injections of C57BL / 6J CFSE + macrophages into C57BL / 6J and with exogenously added APOE3 at 1 , 10 , or 100 μg / ml calculated intraocular concentrations ( n = 6 - 7 / group ; one - way ANOVA / Dunnett test : C57BL / 6J versus 10 μg * P = 0 . 0488 ; C57BL / 6J versus 100 μg ‡ P = 0 . 006 . Mann - Whitney U - test : C57BL / 6J versus 10 μg * P = 0 . 0012 ; C57BL / 6J versus 100 μg ‡ P = 0 . 0013 ) .

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

Figure 4Representative image of a RPE flatmount 12 h after the subretinal injection (red marking) of 4 μl PBS with 12,000 CFSE-stained thioglycollate-elicited peritoneal cells that contain 70% macrophages (inset close-up view).Quantifications of CFSE+F4/80+ macrophages at different time points after subretinal injections of C57BL/6J and Cx3cr1GFP/GFP CFSE+ macrophages (n = 5/per group (12 h) and n = 6/per group thereafter; Mann-Whitney U-test, C57BL/6J versus Cx3cr1GFP/GFP: 1 day n = 20/group *P < 0.0001; 2 day n = 6/group *P = 0.0317).Representative image of TUNEL/Hoechst double-staining 12 h after subretinal injection of C57BL/6J CFSE+ macrophages (experiment repeated three times, inset close-up view).Representative cytometry images of SSC-A/CFSE and CD11b/F4/80 gated analysis of eye cell suspensions prepared 24 h after the injection of Cx3cr1GFP/GFP CFSE+ macrophages and cytometric quantification of eye cell suspensions at 24 h after the injection of C57BL/6J and Cx3cr1GFP/GFP macrophages into C57BL/6J (n = 16-20/group; Mann-Whitney U-test, *P = 0.0024).Quantification of subretinal CFSE+ cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+ magnetic-bead-sorted bone marrow-derived monocytes (Mo) from C57BL/6J and Cx3cr1GFP/GFPmice into C57BL/6Jmice (n = 8-12/group; Mann-Whitney U-test, *P = 0.0006).Quantification of subretinal CFSE+ cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+CD11bFACS-sorted brainmicroglial cells from C57BL/6J and Cx3cr1GFP/GFPmice into C57BL/6Jmice (n = 9-12/group; Mann-Whitney U-test, *P = 0.0087).Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+macrophages from C57BL/6J, Cx3cr1GFP/GFP, Cx3cr1GFP/GFPApoE−/−, and ApoE−/−mice into C57BL/6Jmice (n = 8-12/group; one-way ANOVA/Dunnett test of Cx3cr1GFP/GFP versus any other group *P ≤ 0.0001; Mann-Whitney U-test, Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P = 0.0006).Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injections of C57BL/6J CFSE+ macrophages into C57BL/6J and with exogenously added APOE3 at 1, 10, or 100 μg/ml calculated intraocular concentrations (n = 6-7/group; one-way ANOVA/Dunnett test: C57BL/6J versus 10 μg *P = 0.0488; C57BL/6J versus 100 μg ‡P = 0.006. Mann-Whitney U-test: C57BL/6J versus 10 μg *P = 0.0012; C57BL/6J versus 100 μg ‡P = 0.0013).


Figure 5Quantitative RT - PCR of FasLmRNA normalized with β - actinmRNA of 2 - month - , 12 - month - , and 2 - month - old mice after 4 days of light challenge of C57BL / 6 and Cx3cr1GFP / GFP mouse RPE / choroid plexus ( aging : n = 5 - 6 / group ; Mann - Whitney U - test : 12 months * P = 0 . 0129 ; 4 days light challenge : n = 7 - 8 / group ; * P = 0 . 029 ) . Immunohistochemistry of FASL ( red ) and IBA - 1 ( green ) of a RPE flatmounts and sections ( insets , Hoechst staining blue ) from a 12 - month - old WT ( B ) and Cx3cr1GFP / GFP ( C ) mouse ( representative of three independent experiments , immunostainings omitting the primary antibody served as negative controls ) . Quantitative RT - PCR of FasLmRNA normalized with β - actinmRNA of 12 - month - old C57BL / 6 RPE / choroid plexus extracts 3 h after subretinal injection of C57BL / 6J macrophages or Cx3cr1GFP / GFP macrophages ( n = 6 per group ) , Mann - Whitney U - test , * P = 0 . 0087 . IBA - 1 - stained RPE flatmounts of 2 - month - old C57BL / 6J , Faslgld / gld , and Faslpr / lpr mice after 4 days of light challenge . Quantification of subretinal IBA - 1 + mononuclear phagocytes in control ( left ) and 4 - day light - challenged ( right ) 2 - month - old mice of the indicated strains ( n = 6 - 10 / group ANOVA / Dunnett test at 4 - day light challenge : C57BL / 6J versus FasLgld / gld and C57BL / 6J versus Faslpr / lpr both * P < 0 . 0001 ; Mann - Whitney U - test at 4 - day light challenge : C57BL / 6J versus FasLgld / gld * P < 0 . 0001 ; C57BL / 6J versus Faslpr / lpr * P < 0 . 0001 ) . Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL / 6JCFSE + macrophages into C57BL / 6J and FasLgld / gldmice and Faslpr / lprmacrophages into C57BL / 6Jmice ( n = 10 - 15 / group , one - way ANOVA / Dunnett test : C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus C57BL / 6Jmacrophages inj . into Fasgld / gldmice * P = 0 . 0002 ; C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus Faslpr / lprmacrophages inj . into C57BL / 6Jmice * P < 0 . 0001 . Mann - Whitney U - test : C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus C57BL / 6Jmacrophages inj . into FasLgld / gldmice * P < 0 . 0001 ; C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus Faslpr / lprmacrophages inj . into C57BL / 6Jmice * P < 0 . 0001 ) . Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP / GFP CFSE + macrophages into C57BL / 6J with or without the Fas agonist MegaFasL ( calculated intraocular concentrations 1 ng / ml ; n = 7 - 8 ; Mann - Whitney U - test : * P = 0 . 014 ) . In vitro MegaFasL - induced apoptosis of monocytes and macrophages of the indicated genotypes cultured for 24 h . TUNEL + nucleus quantification expressed as percentage of non - MegaFasL - exposed control .

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 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 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 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 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

Figure 5Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 2-month-, 12-month-, and 2-month-old mice after 4 days of light challenge of C57BL/6 and Cx3cr1GFP/GFP mouse RPE/choroid plexus (aging: n = 5-6/group; Mann-Whitney U-test: 12 months *P = 0.0129; 4 days light challenge: n = 7-8/group; *P = 0.029).Immunohistochemistry of FASL (red) and IBA-1 (green) of a RPE flatmounts and sections (insets, Hoechst staining blue) from a 12-month-old WT (B) and Cx3cr1GFP/GFP(C) mouse (representative of three independent experiments, immunostainings omitting the primary antibody served as negative controls).Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 12-month-old C57BL/6 RPE/choroid plexus extracts 3 h after subretinal injection of C57BL/6J macrophages or Cx3cr1GFP/GFP macrophages (n = 6 per group), Mann-Whitney U-test, *P = 0.0087.IBA-1-stained RPE flatmounts of 2-month-old C57BL/6J, Faslgld/gld, and Faslpr/lpr mice after 4 days of light challenge.Quantification of subretinal IBA-1+ mononuclear phagocytes in control (left) and 4-day light-challenged (right) 2-month-old mice of the indicated strains (n = 6-10/group ANOVA/Dunnett test at 4-day light challenge: C57BL/6J versus FasLgld/gld and C57BL/6J versus Faslpr/lpr both *P < 0.0001; Mann-Whitney U-test at 4-day light challenge: C57BL/6J versus FasLgld/gld *P < 0.0001; C57BL/6J versus Faslpr/lpr *P < 0.0001).Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL/6JCFSE+macrophages into C57BL/6J and FasLgld/gldmice and Faslpr/lprmacrophages into C57BL/6Jmice (n = 10-15/group, one-way ANOVA/Dunnett test: C57BL/6Jmacrophages inj. into C57BL/6Jmice versus C57BL/6Jmacrophages inj. into Fasgld/gldmice *P = 0.0002; C57BL/6Jmacrophages inj. into C57BL/6Jmice versus Faslpr/lprmacrophages inj. into C57BL/6Jmice *P < 0.0001. Mann-Whitney U-test: C57BL/6Jmacrophages inj. into C57BL/6Jmice versus C57BL/6Jmacrophages inj. into FasLgld/gldmice *P < 0.0001; C57BL/6Jmacrophages inj. into C57BL/6Jmice versus Faslpr/lprmacrophages inj. into C57BL/6Jmice *P < 0.0001).Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP/GFP CFSE+ macrophages into C57BL/6J with or without the Fas agonist MegaFasL (calculated intraocular concentrations 1 ng/ml; n = 7-8; Mann-Whitney U-test: *P = 0.014).In vitro MegaFasL-induced apoptosis of monocytes and macrophages of the indicated genotypes cultured for 24 h. TUNEL+ nucleus quantification expressed as percentage of non-MegaFasL-exposed control.


Figure 6Quantitative RT - PCR of FasLmRNA normalized with β - actinmRNA of 12 - month - old C57BL / 6 RPE / choroid plexus 3 h after subretinal injection of PBS ( PBS ; n = 36 ) , IL - 6 ( n = 17 ) , and APOE3 ( n = 21 ) ; calculated intraocular concentrations : 5 ng / ml and 10 μg / ml , respectively ; one - way ANOVA / Dunnett post hoc test PBS versus IL - 6 * P = 0 . 038 ; t - test PBS versus IL - 6 * P = 0 . 043 . Mouse IL - 6 ELISA of supernatants from C57BL / 6J peritoneal macrophages incubated for 24 h in control medium , lipid - free APOE3 ( 5 μg / ml ) , APOE3 ( 5 μg / ml ) , and polymyxin B ( 25 μg / ml ) , heat - denatured APOE3 ( dAPOE3 , 5 μg / ml ) , APOE3 ( 5 μg / ml ) , and rat IgG1 isotype control ( IgG , 100 μg / ml ) or APOE3 ( 5 μg / ml ) and rat anti - CD14 antibody ( aCD14 Ab , 100 μg / ml ) or APOE3 ( 5 μg / ml ) and rat anti - TLR2 antibody ( aTLR2 Ab , 100 μg / ml ) ( n = 5 - 6 / group ; one - way ANOVA / Bonferroni multi - comparison tests : APOE3 versus CTL * P < 0 . 0001 ; dAPOE3 versus APOE3 # P < 0 . 0001 ; APOE3 IgG versus CTL P < 0 . 0001 ; APOE3 IgG versus APOE3 aCD14 Ab § P < 0 . 0001 ; APOE3 IgG versus APOE3 aTLR2 Ab + + P < 0 . 0001 . Mann - Whitney U - test : APOE3 versus CTL * P = 0 . 0043 ; dAPOE3 versus APOE3 # P = 0 . 0117 ; APOE3 IgG versus CTL P = 0 . 0080 ; APOE3 IgG versus APOE3 aCD14 Ab § P = 0 . 0117 ; APOE3 IgG versus APOE3 aTLR2 Ab + + P = 0 . 0079 . The experiment was repeated twice with similar results ) . Quantitative RT - PCR of IL - 6mRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP , and Cx3cr1GFP / GFP ApoE − / − peritoneal macrophages cultured for 24 h with CX3CL1 ( n = 5 per group , one - way ANOVA / Bonferroni post hoc multi - comparison tests : C57BL / 6J versus Cx3cr1GFP / GFP * P < 0 . 0001 and Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − ‡ P < 0 . 0001 . Mann - Whitney U - test : C57BL / 6J versus Cx3cr1GFP / GFP * P = 0 . 0079 and Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − ‡ P = 0 . 0079 . The experiment was repeated twice with similar results . ) . Orthogonal and lateral Z - stack projection of IL - 6 ( D , red ) , IBA - 1 + ( E , green ) , and phalloidin ( E , blue ) of IBA + mononuclear phagocytes adjacent to the phalloidin + RPE of a retinal flatmount from a 12 - month - old Cx3cr1GFP / GFP mouse . ( Representative of three independent experiments , immunostainings omitting the primary antibody served as negative controls ) . Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL / 6J CFSE + macrophages into C57BL / 6J with or without IL - 6 ( n = 15 - 20 / group ; calculated intraocular concentrations of 5 ng / ml . Mann - Whitney U - test : * P < 0 . 0001 ) . Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP / GFPCFSE + Mϕs into C57BL / 6J with control ( IgG , n = 16 ) or anti - IL - 6 antibody ( aIL - 6 Ab , n = 8 ; calculated intraocular concentrations 5 μg / ml ; per group . Mann - Whitney U - test : * P = 0 . 0036 ) . 7 day laser - injured IBA - 1 ( green ) and CD102 ( red ) double - stained RPE flatmounts of control IgG - ( I ) and anti - IL - 6 - treated ( J ) Cx3cr1GFP / GFP mice . Quantification of subretinal IBA - 1 + mononuclear phagocytes / impact localized on the lesion surrounding RPE of Cx3cr1GFP / GFP mice treated with control IgG , IL - 6 - , or CD14 - blocking antibodies ( calculated intraocular concentration 5 μg / ml ; n = 13 - 14 / group , one - way ANOVA / Dunnett ' s post hoc tests of IgG versus any other group * P < 0 . 001 . Mann - Whitney U - test IgG versus anti - IL - 6 * P = 0 . 0021 ; IgG versus anti CD14 * P = 0 . 0028 ) .

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

Figure 6Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 12-month-old C57BL/6 RPE/choroid plexus 3 h after subretinal injection of PBS (PBS; n = 36), IL-6 (n = 17), and APOE3 (n = 21); calculated intraocular concentrations: 5 ng/ml and 10 μg/ml, respectively; one-way ANOVA/Dunnett post hoc test PBS versus IL-6 *P = 0.038; t-test PBS versus IL-6 *P = 0.043.Mouse IL-6 ELISA of supernatants from C57BL/6J peritoneal macrophages incubated for 24 h in control medium, lipid-free APOE3 (5 μg/ml), APOE3 (5 μg/ml), and polymyxin B (25 μg/ml), heat-denatured APOE3 (dAPOE3, 5 μg/ml), APOE3 (5 μg/ml), and rat IgG1 isotype control (IgG, 100 μg/ml) or APOE3 (5 μg/ml) and rat anti-CD14 antibody (aCD14 Ab, 100 μg/ml) or APOE3 (5 μg/ml) and rat anti-TLR2 antibody (aTLR2 Ab, 100 μg/ml) (n = 5-6/group; one-way ANOVA/Bonferroni multi-comparison tests: APOE3 versus CTL *P < 0.0001; dAPOE3 versus APOE3 #P < 0.0001; APOE3 IgG versus CTL P < 0.0001; APOE3 IgG versus APOE3 aCD14 Ab §P < 0.0001; APOE3 IgG versus APOE3 aTLR2 Ab ++P <0.0001. Mann-Whitney U-test: APOE3 versus CTL *P = 0.0043; dAPOE3 versus APOE3 #P = 0.0117; APOE3 IgG versus CTL P = 0.0080; APOE3 IgG versus APOE3 aCD14 Ab §P = 0.0117; APOE3 IgG versus APOE3 aTLR2 Ab ++P = 0.0079. The experiment was repeated twice with similar results).Quantitative RT-PCR of IL-6mRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP, and Cx3cr1GFP/GFP ApoE−/− peritoneal macrophages cultured for 24 h with CX3CL1 (n = 5 per group, one-way ANOVA/Bonferroni post hoc multi-comparison tests: C57BL/6J versus Cx3cr1GFP/GFP *P < 0.0001 and Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/−‡P < 0.0001. Mann-Whitney U-test: C57BL/6J versus Cx3cr1GFP/GFP *P = 0.0079 and Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/−‡P = 0.0079. The experiment was repeated twice with similar results.).Orthogonal and lateral Z-stack projection of IL-6 (D, red), IBA-1+ (E, green), and phalloidin (E, blue) of IBA+ mononuclear phagocytes adjacent to the phalloidin+ RPE of a retinal flatmount from a 12-month-old Cx3cr1GFP/GFP mouse. (Representative of three independent experiments, immunostainings omitting the primary antibody served as negative controls).Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL/6J CFSE+ macrophages into C57BL/6J with or without IL-6 (n = 15-20/group; calculated intraocular concentrations of 5 ng/ml. Mann-Whitney U-test: *P < 0.0001).Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP/GFPCFSE+Mϕs into C57BL/6J with control (IgG, n = 16) or anti-IL-6 antibody (aIL-6 Ab, n = 8; calculated intraocular concentrations 5 μg/ml; per group. Mann-Whitney U-test: *P = 0.0036).7 day laser-injured IBA-1 (green) and CD102 (red) double-stained RPE flatmounts of control IgG- (I) and anti-IL-6-treated (J) Cx3cr1GFP/GFP mice.Quantification of subretinal IBA-1+ mononuclear phagocytes/impact localized on the lesion surrounding RPE of Cx3cr1GFP/GFP mice treated with control IgG, IL-6-, or CD14-blocking antibodies (calculated intraocular concentration 5 μg/ml; n = 13-14/group, one-way ANOVA/Dunnett's post hoc tests of IgG versus any other group *P < 0.001. Mann-Whitney U-test IgG versus anti-IL-6 *P = 0.0021; IgG versus anti CD14*P = 0.0028).


Figure 1 A Neonatal rat ventricular myocytes ( NRVMs ) were infected with the 3xMEF2 - Luc reporter , serum starved for 20 hours and stimulated for 24 hours with different agonists : 100 nM endothelin - 1 ( ET1 ) , 1 μM sphingosine - 1 - phosphate ( S1P ) , 10 μM lisophosphatidic acid ( LPA ) , 1 μM WIN55 , 212 - 2 ( WIN55 , cannabinoid receptor agonist ) , 1 μM isoproterenol ( ISO , β - adrenergic receptor agonist ) , 100 nM angiotensin II ( AngII ) , 10 μM prostaglandin E1 ( PGE1 ) , 10 μM prostaglandin E2 ( PGE2 ) or 100 nM prostanoid F receptor agonist fluprostenol ( Flupro ) , 100 nM treprostinil ( Trepro , prostacyclin receptor agonist ) Data information : values are mean ± s . e . m . In ( A ) , the experiment was performed in triplicates , similar results were obtained in 3 different experiments . Student ' s two - tailed t - test , * , P < 0 . 05 vs . control ( ET1 , P = 0 . 0013 ; S1P , P = 0 . 0144 ; LPA , P = 0 . 026 ; WIN55 , P = 0 . 6205 ; ISO = 0 . 0399 ; AngII , P = 0 . 5812 ; PGE1 , P = 0 . 001 ; PGE2 , P < 0 . 0001 ; Flupro , P = 0 . 0846 ; Trepro , P = 0 . 1958 ) B PGE2 induces the expression of MEF2 target genes . NRVMs were serum - starved for 24 hours , then were stimulated with 1 μM PGE2 for 2 hours and mRNA levels of the indicated genes were determined Data information : values are mean ± s . e . m In ( B ) , n = 5 , technical replicates , Student ' s two - tailed t - test , * , P < 0 . 05 vs . control ( Nur77 , P = 0 . 0148 ; Myomaxin , P = 0 . 0417 ; Adamts1 , P = 0 . 0112 ; BNP , P = 0 . 0065 ) C Correlation between MEF2 activation and NRVM hypertrophy . MEF2 activity was measured as in ( A ) upon increasing concentration of PGE2 . Cell size of NRVMs was determined after 48 - hour stimulation with the indicated concentrations of PGE2 . Left : representative images of α - actinin stained NRVMs stimulated with DMSO , 300 nM PGE2 , or 1 nM ET1 . Scale bar is 10 µm Data information : values are mean ± s . e . m In ( C ) , NRVM cell size was determined from a minimum of 100 cells of 3 technical replicates upon different concentrations of PGE2 , MEF2 - activity was parallel assessed from 6 technical replicates . Correlation was statistically analyzed by calculating Pearson correlation coefficient ( Pearson r = 0 . 9626 , P = 0 . 0021 ) D , PGE2 activates MEF2 via EP3 receptor . NRVMs were infected with the 3xMEF2 - Luc reporter and serum - starved for 20 hours . The cells were stimulated with DMSO or 1 μM PGE2 for 24 hours in the presence or absence of different EP receptor antagonists : AH6809 ( 10 μM , EP1 - and EP2 - antagonist ) or 798106 ( 200 nM , EP3 - antagonist ) or L161 , 982 ( 2 μM , EP4 - antagonist ) Data information : values are mean ± s . e . m In ( D ) , n = 3 , independent experiments , * represents significant interaction between the two treatments ( P < 0 . 05 , Two - Way ANOVA , AH6809 , P = 0 . 1092 ; L798106 , P = 0 . 0002 ; L161 , 982 , P = 0 . 5579 ) . The exact n and P values can be also found in the Source Data excel file for Figure

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Neonatal rat ventricular myocytes (NRVMs) were infected with the 3xMEF2-Luc reporter, serum starved for 20 hours and stimulated for 24 hours with different agonists: 100 nM endothelin-1 (ET1), 1 μM sphingosine-1-phosphate (S1P), 10 μM lisophosphatidic acid (LPA), 1 μM WIN55,212-2 (WIN55, cannabinoid receptor agonist), 1 μM isoproterenol (ISO, β-adrenergic receptor agonist), 100 nM angiotensin II (AngII), 10 μM prostaglandin E1 (PGE1), 10 μM prostaglandin E2 (PGE2) or 100 nM prostanoid F receptor agonist fluprostenol (Flupro), 100 nM treprostinil (Trepro, prostacyclin receptor agonist) Data information: values are mean±s.e.m. In (A), the experiment was performed in triplicates, similar results were obtained in 3 different experiments. Student's two-tailed t-test, *,P<0.05 vs. control (ET1, P=0.0013; S1P, P=0.0144; LPA, P=0.026; WIN55, P=0.6205; ISO=0.0399; AngII, P=0.5812; PGE1, P=0.001; PGE2, P<0.0001; Flupro, P=0.0846; Trepro, P=0.1958)  B PGE2 induces the expression of MEF2 target genes. NRVMs were serum-starved for 24 hours, then were stimulated with 1 μM PGE2 for 2 hours and mRNA levels of the indicated genes were determined Data information: values are mean±s.e.m In (B), n=5, technical replicates, Student's two-tailed t-test, *,P<0.05 vs. control (Nur77, P=0.0148; Myomaxin, P=0.0417; Adamts1, P=0.0112; BNP, P=0.0065) C Correlation between MEF2 activation and NRVM hypertrophy. MEF2 activity was measured as in (A) upon increasing concentration of PGE2. Cell size of NRVMs was determined after 48-hour stimulation with the indicated concentrations of PGE2. Left: representative images of α-actinin stained NRVMs stimulated with DMSO, 300 nM PGE2, or 1 nM ET1. Scale bar is 10 µm Data information: values are mean±s.e.m In (C), NRVM cell size was determined from a minimum of 100 cells of 3 technical replicates upon different concentrations of PGE2, MEF2-activity was parallel assessed from 6 technical replicates. Correlation was statistically analyzed by calculating Pearson correlation coefficient (Pearson r=0.9626, P=0.0021) D, PGE2 activates MEF2 via EP3 receptor. NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were stimulated with DMSO or 1 μM PGE2 for 24 hours in the presence or absence of different EP receptor antagonists: AH6809 (10 μM, EP1- and EP2-antagonist) or 798106 (200 nM, EP3-antagonist) or L161,982 (2 μM, EP4-antagonist) Data information: values are mean±s.e.m In (D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, AH6809, P=0.1092; L798106, P=0.0002; L161,982, P=0.5579). The exact n and P values can be also found in the Source Data excel file for Figure 1


Figure 2 B - D ( B , D ) NRVMs were infected with the 3xMEF2 - Luciferase reporter and with recombinant adenoviruses encoding EGFP ( AdGFP ) , RGS16 , RGS2 , p63ΔN , RGS - LSCII , or Gαt , as indicated . The cells were serum starved for 20 hours and stimulated with DMSO or 1 μM PGE2 for 24 hours . ( C ) NRVMs were infected with the 3xMEF2 - Luciferase reporter and pretreated with the adenylyl cyclase inhibitor SQ22536 ( 100 μM ) for 20 minutes or the Gi / o - protein inhibitor pertussis toxin ( PTX , 100 ng / ml ) for 20 hours in serum - starved conditions and treated with DMSO or 1 μM PGE2 for 24 hours . Data information : In ( B - D ) , n = 3 , independent experiments , * represents significant interaction between the two treatments ( P < 0 . 05 , Two - Way ANOVA , RGS16 , P < 0 . 0001 ; RGS2 , P = 0 . 1725 ; p63ΔN , P = 0 . 0666 ; RGS - LSCII , P = 0 . 0818 , SQ22536 , P = 0 , 385 ; PTX , P < 0 . 0001 ; Gαt , P < 0 , 0001 ) , values are mean ± s . e . m . The exact n and P values can be also found in the Source Data excel file for Figure 2

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-D (B, D) NRVMs were infected with the 3xMEF2-Luciferase reporter and with recombinant adenoviruses encoding EGFP (AdGFP), RGS16, RGS2, p63ΔN, RGS-LSCII, or Gαt, as indicated. The cells were serum starved for 20 hours and stimulated with DMSO or 1 μM PGE2 for 24 hours. (C) NRVMs were infected with the 3xMEF2-Luciferase reporter and pretreated with the adenylyl cyclase inhibitor SQ22536 (100 μM) for 20 minutes or the Gi/o-protein inhibitor pertussis toxin (PTX, 100 ng/ml) for 20 hours in serum-starved conditions and treated with DMSO or 1 μM PGE2 for 24 hours. Data information: In (B-D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, RGS16, P<0.0001; RGS2, P=0.1725; p63ΔN, P=0.0666; RGS-LSCII, P=0.0818, SQ22536, P=0,385; PTX, P<0.0001; Gαt, P<0,0001), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 2


Figure 3 A , B NRVMs were infected with recombinant adenovirus encoding Flag - HDAC5 ( A ) or not infected ( B ) and serum starved for 20 hours . The cells were pretreated with the PKC - inhibitor bisindolylmaleimide I ( 2 μM ) or the PKD - inhibitor BPKDi ( 3 μM ) for 20 minutes and stimulated with 100 nM endothelin - 1 ( ET1 ) or 100 μM phenylephrine ( PE ) or 1 μM PGE2 for 4 hours , as indicated . HDAC phosphorylation was detected by immunoblotting with the anti - HDAC5 phospho - Ser - 498 antibody , phosphorylation of PKD was assessed using anti - phospho - PKD antibodies ( Ser - 744 / Ser - 748 and Ser - 916 ) , representative images are shown from 3 independent experiments C NRVMs were infected with the 3xMEF2 - Luc reporter and serum - starved for 20 hours . The cells were pretreated with the PKD - inhibitor BPKDi ( 3 μM ) or the CamKII - inhibitor AIP ( 1 μM ) for 20 minutes and stimulated with DMSO or 1 μM PGE2 for 24 hours . N = 3 , * represents significant interaction between the two treatments ( P < 0 . 05 , Two - Way ANOVA , BPKDi , P < 0 . 0001 ; AIP , P = 0 . 2197 ) , values are mean ± s . e . m . The exact n and P values can be also found in the Source Data excel file for Figure

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,B NRVMs were infected with recombinant adenovirus encoding Flag-HDAC5 (A) or not infected (B) and serum starved for 20 hours. The cells were pretreated with the PKC-inhibitor bisindolylmaleimide I (2 μM) or the PKD-inhibitor BPKDi (3 μM) for 20 minutes and stimulated with 100 nM endothelin-1 (ET1) or 100 μM phenylephrine (PE) or 1 μM PGE2 for 4 hours, as indicated. HDAC phosphorylation was detected by immunoblotting with the anti-HDAC5 phospho-Ser-498 antibody, phosphorylation of PKD was assessed using anti-phospho-PKD antibodies (Ser-744/Ser-748 and Ser-916), representative images are shown from 3 independent experiments  C NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were pretreated with the PKD-inhibitor BPKDi (3 μM) or the CamKII-inhibitor AIP (1 μM) for 20 minutes and stimulated with DMSO or 1 μM PGE2 for 24 hours. N=3, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, BPKDi, P<0.0001; AIP, P=0.2197), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 3


Figure 4 A NRVMs were serum - starved for 24 hours and stimulated for 2 minutes with 10 μM PGE2 . Rac1 and RhoA GTPase activation was determined by effector pulldown assay and analyzed by immunoblot Data information : In ( A - C ) representative images from at least 3 independent experiments are shown B NRVMs were infected with recombinant adenoviruses encoding EGFP ( AdGFP ) or RGS16 24 hours prior to stimulation , then Rac1 activity was measure Data information : In ( A - C ) representative images from at least 3 independent experiments are shown . In ( B - D ) values are mean ± s . e . m . , ( B , C ) n = 4 3 ; * , P < 0 . 05 ( Two - Way ANOVA , RGS16 , P = 0 . 0415 ( B ) , NSC effect on Rac1 activation , P = 0 . 0412 ( C ) ; NSC effect on MEF2 induction , P < 0 . 0001 . PGE2 effect was significant in Rac1 activation , P < 0 . 0001 ) . The exact n and P values can be also found in the Source Data excel file for Figure 4 C Cells were pretreated with 100 ng / ml pertussis toxin ( PTX , Gi / o - inhibitor ) for 24 hours or with 50 μM NSC23766 ( NSC , Tiam1 - Rac1 interaction inhibitor ) for 2 hours . Thereafter Rac1 activation was determined by effector pulldown assay Data information : In ( A - C ) representative images from at least 3 independent experiments are shown In ( B - D ) values are mean ± s . e . m . , ( B , C ) n = 4 3 ; * , P < 0 . 05 ( Two - Way ANOVA , RGS1 PTX , P = 0 . 039 ( C ) , NSC effect on Rac1 activation , P = 0 . 0412 ( C ) ; NSC effect on MEF2 induction , P < 0 . 0001 . PGE2 effect was significant in Rac1 activation , P < 0 . 0001 ) . The exact n and P values can be also found in the Source Data excel file for Figure 4 D NRVMs were transduced with the 3xMEF2 - Luc reporter and were serum - starved for 20 hours . After 2 - hour of 50 μM NSC23766 ( A ) pretreatments , the cells were stimulated with DMSO or 1 μM PGE2 for 24 hours , then MEF2 activity was determined . NSC abolished the MEF2 activation by PGE2 Data information In ( B - D ) values are mean ± s . e . m . ( D ) , n = 3 ; * , P < 0 . 05 ( Two - Way ANOVA ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 NRVMs were serum-starved for 24 hours and stimulated for 2 minutes with 10 μM PGE2. Rac1 and RhoA GTPase activation was determined by effector pulldown assay and analyzed by immunoblot Data information: In (A-C) representative images from at least 3 independent experiments are shown  B NRVMs were infected with recombinant adenoviruses encoding EGFP (AdGFP) or RGS16 24 hours prior to stimulation, then Rac1 activity was measure Data information: In (A-C) representative images from at least 3 independent experiments are shown. In (B-D) values are mean±s.e.m., (B,C) n=4 3; *,P<0.05 (Two-Way ANOVA, RGS16, P=0.0415 (B ), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4 C Cells were pretreated with 100 ng/ml pertussis toxin (PTX, Gi/o-inhibitor) for 24 hours or with 50 μM NSC23766 (NSC, Tiam1-Rac1 interaction inhibitor) for 2 hours. Thereafter Rac1 activation was determined by effector pulldown assay Data information: In (A-C) representative images from at least 3 independent experiments are shown In (B-D) values are mean±s.e.m., (B,C) n=4 3; *,P<0.05 (Two-Way ANOVA, RGS1 PTX, P=0.039 (C), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4 D NRVMs were transduced with the 3xMEF2-Luc reporter and were serum-starved for 20 hours. After 2-hour of 50 μM NSC23766 (A) pretreatments, the cells were stimulated with DMSO or 1 μM PGE2 for 24 hours, then MEF2 activity was determined. NSC abolished the MEF2 activation by PGE2 Data information In (B-D) values are mean±s.e.m. (D), n=3; *,P<0.05 (Two-Way ANOVA, RGS1


Figure 5 A After serum starvation NRVMs were pretreated with or without 50 μM NSC23766 for 2 hours and stimulated with 1 μM PGE2 for 1 hour . Total and phosphorylated PKD levels were analyzed by immunoblot . PKD was activated by PGE2 , but it was not affected by NSC Data information : In ( A - C ) representative images from 3 independent experiments are shown B After infection with Flag - HDAC5 and serum starvation , NRVMs were treated with 50 μM NSC23766 for 2 hours prior to stimulation . The cells were stimulated with 1 μM PGE2 for 1 hour . HDAC5 phosphorylation at Ser - 498 was detected by immunoblot . The phosphorylation of HDAC5 at this site was not prevented by NSC Data information : In ( A - C ) representative images from 3 independent experiments are shown C NRVMs and neonatal cardiac fibroblasts ( CFs ) were serum - starved , pretreated with vehicle or 200 nM L798106 ( EP3 receptor antagonist ) for 20 minutes , and were stimulated with 1 μM PGE2 for 10 minutes . PAK2 and PKD phosphorylation was assessed by immunoblot . Calsequestrin was used as a myocyte - marker Data information : In ( A - C ) representative images from 3 independent experiments are shown D MEF2 activity was determined in cells infected with the 3xMEF2 - Luc reporter after serum starvation . Cells were stimulated with vehicle or 1 μM PGE2 for 24 hours after 1 - hour of 30 μM IPA - 3 pretreatment . IPA - 3 inhibited the MEF2 activation by PGE2 Data information In ( D ) values are mean ± s . e . m . , n = 3 , biological replicates , * , P < 0 . 05 ( two - way ANOVA , P < 0 . 0001 ) . The exact n and P values can be also found in the Source Data excel file for Figure

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5 A After serum starvation NRVMs were pretreated with or without 50 μM NSC23766 for 2 hours and stimulated with 1 μM PGE2 for 1 hour. Total and phosphorylated PKD levels were analyzed by immunoblot. PKD was activated by PGE2, but it was not affected by NSC Data information: In (A-C) representative images from 3 independent experiments are shown  B After infection with Flag-HDAC5 and serum starvation, NRVMs were treated with 50 μM NSC23766 for 2 hours prior to stimulation. The cells were stimulated with 1 μM PGE2 for 1 hour. HDAC5 phosphorylation at Ser-498 was detected by immunoblot. The phosphorylation of HDAC5 at this site was not prevented by NSC Data information: In (A-C) representative images from 3 independent experiments are shown  C NRVMs and neonatal cardiac fibroblasts (CFs) were serum-starved, pretreated with vehicle or 200 nM L798106 (EP3 receptor antagonist) for 20 minutes, and were stimulated with 1 μM PGE2 for 10 minutes. PAK2 and PKD phosphorylation was assessed by immunoblot. Calsequestrin was used as a myocyte-marker Data information: In (A-C) representative images from 3 independent experiments are shown  D MEF2 activity was determined in cells infected with the 3xMEF2-Luc reporter after serum starvation. Cells were stimulated with vehicle or 1 μM PGE2 for 24 hours after 1-hour of 30 μM IPA-3 pretreatment. IPA-3 inhibited the MEF2 activation by PGE2 Data information In (D) values are mean±s.e.m., n=3, biological replicates, *,P<0.05 (two-way ANOVA, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 5


Figure 6 Top : GFP - HDAC5 infected NRVMs were serum - starved for a day , pretreated for two hours with the indicated compounds ( BPKDi , 3 µM , IPA - 3 , 30 µM ) alone or in combination , then were stimulated for 4 hours . Representative images . Scale bar is 10 µm . Bottom : Statistical analysis of cellular localization of HDAC5 . GFP - HDAC5 localization ( nuclear or cytosolic ) was assessed in > 100 cells of each sample ( n = 3 , technical replicates ) . * , P < 0 , 05 ( one - way ANOVA with Bonferroni post - hoc test , vehicle vs . ET1 or vs . PGE2 , P < 0 . 0001 ; PGE2 vs . PGE2 + BPKDi , P = 0 . 0004 ; PGE2 vs . PGE2 + BPKDi , P = 0 . 0114 ; PGE2 vs . PGE2 + BPKDi + IPA - 3 , P < 0 . 0001 ) . The exact n and P values can be also found in the Source Data excel file for Figure 6

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6 Top: GFP-HDAC5 infected NRVMs were serum-starved for a day, pretreated for two hours with the indicated compounds (BPKDi, 3 µM, IPA-3, 30 µM) alone or in combination, then were stimulated for 4 hours. Representative images. Scale bar is 10 µm. Bottom : Statistical analysis of cellular localization of HDAC5. GFP-HDAC5 localization (nuclear or cytosolic) was assessed in >100 cells of each sample (n=3, technical replicates). *, P<0,05 (one-way ANOVA with Bonferroni post-hoc test, vehicle vs. ET1 or vs. PGE2, P<0.0001; PGE2 vs. PGE2+BPKDi, P=0.0004; PGE2 vs. PGE2+BPKDi, P=0.0114; PGE2 vs. PGE2+BPKDi+IPA-3, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 6


Figure 7 MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 weeks old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally and were sacrificed after 24 hours A mRNA - levels of different inflammatory cytokines , as indicated . The graphs show relative mRNA - levels , fold increase compared to saline - treated controls , normalized to 18s - content Data information : values are mean ± s . e . m . The exact n values are shown in the bottom of the bar graphs . * , P < 0 . 05 , Welch ' s two - tailed unpaired t - test was used for statistical analysis , n . s . = not significant P values : IL6 , P = 0 . 0007 ( A ) ; TNFα , P < 0 . 0001 ( A ) MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 weeks old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally and were sacrificed after 24 hours B PGE2 was quantified after mechanical homogenization of deeply frozen hearts using nano - liquid chromatography tandem mass spectrometry Data information : values are mean ± s . e . m . The exact n values are shown in the bottom of the bar graphs . * , P < 0 . 05 , Welch ' s two - tailed unpaired t - test was used for statistical analysis , n . s . = not significant P values PGE2 , P = 0 . 0016 ( B MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 weeks old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally and were sacrificed after 24 hours C Histologic and macroscopic stainings of β - galactosidase - activity in MEF2 - lacZ reporter mice show MEF2 - activation ( blue cells and precipitates , respectively ) in the myocardium upon LPS - treatment . Saline - treated littermates served as control . Scale bar of histological stainings is 100 µm . Quantification of whole - heart stainings is shown in the right panel ( pixel intensity in blue channel normalized to total intensity ) Data information : values are mean ± s . e . m . The exact n values are shown in the bottom of the bar graphs . * , P < 0 . 05 , Welch ' s two - tailed unpaired t - test was used for statistical analysis , n . s . = not significant . In ( C ) representative images of myocardial sections are shown from 3 independent experiments and representative whole hearts of 6 independent experiments . P values blue pixel intensity , P = 0 . 0083 ( C ) MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 weeks old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally and were sacrificed after 24 hours D Induction of PKD , HDAC5 and PAK2 in myocardial inflammations . Immunoblots were performed on extracts of hearts of saline - and LPS - treated mice . Representative blots are shown in the left panel , the quantification is in the right panel Data information : values are mean ± s . e . m The exact n values are shown in the bottom of the bar graphs . * , P < 0 . 05 , Welch ' s two - tailed unpaired t - test was used for statistical analysis , n . s . = not significant P values ) ; phospho - PKD - Ser - 744 / Ser - 748 , P = 0 . 0049 ( D ) ; phospho - PKD - Ser - 916 , P = 0 . 2684 ( D ) ; phospho - HDAC5 - Ser - 498 ( D ) , P = 0 . 0019 ; phospho - HDAC5 - Ser - 259 , P = 0 . 0003 ( D ) ; phospho - PAK2 - Ser - 192 / Ser - 197 , P = 0 . 1962 ( D ) ; phospho - PAK2 - Thr - 402 , P = 0 . 0446 ( D ) . The exact n and P values can be also found in the Source Data excel file for Figure

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7 MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours A mRNA-levels of different inflammatory cytokines, as indicated. The graphs show relative mRNA-levels, fold increase compared to saline-treated controls, normalized to 18s-content Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values: IL6, P=0.0007 (A); TNFα, P<0.0001 (A) MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours B PGE2 was quantified after mechanical homogenization of deeply frozen hearts using nano-liquid chromatography tandem mass spectrometry Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values PGE2, P=0.0016 (B MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours C Histologic and macroscopic stainings of β-galactosidase-activity in MEF2-lacZ reporter mice show MEF2-activation (blue cells and precipitates, respectively) in the myocardium upon LPS-treatment. Saline-treated littermates served as control. Scale bar of histological stainings is 100 µm. Quantification of whole-heart stainings is shown in the right panel (pixel intensity in blue channel normalized to total intensity) Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant. In (C) representative images of myocardial sections are shown from 3 independent experiments and representative whole hearts of 6 independent experiments. P values blue pixel intensity, P=0.0083 (C) MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours D Induction of PKD, HDAC5 and PAK2 in myocardial inflammations. Immunoblots were performed on extracts of hearts of saline- and LPS-treated mice. Representative blots are shown in the left panel, the quantification is in the right panel Data information: values are mean±s.e.m The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values ); phospho-PKD-Ser-744/Ser-748, P=0.0049 (D); phospho-PKD-Ser-916, P=0.2684 (D); phospho-HDAC5-Ser-498 (D), P=0.0019; phospho-HDAC5-Ser-259, P=0.0003 (D); phospho-PAK2-Ser-192/Ser-197, P=0.1962 (D); phospho-PAK2-Thr-402, P=0.0446 (D). The exact n and P values can be also found in the Source Data excel file for Figure 7