I Basal and stimulated ( + 100 nM isoproterenol ) lipolysis , as measured by glycerol ( left ) and fatty acids ( right ) released from explants of epididymal WAT from overnight fasted 10 - week - old male WT and FADD - D mice fed a HFD ( n = 4 for each genotype ) . Data are expressed as mean ± SEM . * P = 0 . 0078 , * * P = 0 . 0065 , * * * P = 0 . 0008 , ^ P = 0 . 0133 , ^ ^ P = 0 . 0053 , ^ ^ ^ P = 0 . 0006 , ^ ^ ^ ^ P = 0 . 0001 , # P = 0 . 0082 , # # P = 0 . 0051 , # # # P = 0 . 0009 , * ^ P = 0 . 0151 , * ^ ^ P = 0 . 0019 , * * ^ ^ P = 0 . 0005 , * * * ^ P = 0 . 0002 ( Student ' s t - test ) .

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

I Basal and stimulated (+ 100 nM isoproterenol) lipolysis, as measured by glycerol (left) and fatty acids (right) released from explants of epididymal WAT from overnight fasted 10-week-old male WT and FADD-D mice fed a HFD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0078, **P = 0.0065, ***P = 0.0008, ^P = 0.0133, ^^P = 0.0053, ^^^P = 0.0006, ^^^^P = 0.0001, #P = 0.0082, ##P = 0.0051, ###P = 0.0009, *^P = 0.0151, *^^P = 0.0019, **^^P = 0.0005, ***^P = 0.0002 (Student's t-test).


J Molar ratio of FFA to glycerol release from WAT explants ( n = 4 for each genotype ) . Data are expressed as mean ± SEM . * P = 0 . 0412 , * * P = 0 . 0033 ( Student ' s t - test ) .

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

J Molar ratio of FFA to glycerol release from WAT explants (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0412, **P = 0.0033 (Student's t-test).


A 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 .

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

A 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 ) .

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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 ) .

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

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 .

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

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 .

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

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 .

O O O O O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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).


A 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 ) .

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

A 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 ) .

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

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 ) .

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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 ) .

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

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

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.


A - 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 .

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

A-C 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 ) .

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

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 ) .

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

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

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).


A 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 ) .

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

A 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 ) .

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

C 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 .

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

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 ) .

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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 ) .

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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 ) .

O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-CELL_LINE O O O B-CELL_TYPE O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A 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 ) .

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

A 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 ) .

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

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 ) .

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

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 .

O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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

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).


A 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 .

O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O B-TISSUE O B-TISSUE O

A 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 ) .

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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 ) .

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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 ) .

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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 ) .

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

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 ) .

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

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 .

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

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 ) .

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

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 .

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

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

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.


( C ) Figures show the presence of differentially expressed genes within the analyzed single cells . Black color indicates cells with RPKM values above 40 for Pax3 , 300 for Stmn2 , 200 for Slc1a3 and 100 for

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

(C) Figures show the presence of differentially expressed genes within the analyzed single cells. Black color indicates cells with RPKM values above 40 for Pax3, 300 for Stmn2, 200 for Slc1a3 and 100 for Mbp


( B ) Expression ( RPKM ) of Sox3 and Sox9 in NPC and GPC cultures from RNA - seq experiments . Results are represented as mean ± SEM from 3 - 4 experiments ( n = 3 for NPC and n = 4 for GPC , * * * equals p = 4x10 - 5 , t . test )

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(B) Expression (RPKM) of Sox3 and Sox9 in NPC and GPC cultures from RNA-seq experiments. Results are represented as mean ± SEM from 3-4 experiments (n=3 for NPC and n=4 for GPC, *** equals p=4x10-5, t.test)


( C ) Immunohistochemical analysis of SOX3 and SOX9 expression in ESC derived NPC and GPC cultures . Analysis of TUJ1 , GFAP and SOX10 protein expression in differentiated cultures revealed that NPC give rise to neurons and GPCs to astrocytes and oligodendrocytes . Scale bar represents

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(C) Immunohistochemical analysis of SOX3 and SOX9 expression in ESC derived NPC and GPC cultures. Analysis of TUJ1, GFAP and SOX10 protein expression in differentiated cultures revealed that NPC give rise to neurons and GPCs to astrocytes and oligodendrocytes. Scale bar represents 40µm


1 ] . ( F ) H3K27Ac read density in NPCs or GPCs around DNA - regions bound by SOX3 in NPCs . SOX3 DNA - regions with different H3K27Ac read density patterns in NPCs and GPCs were categorized as group I and II loci . Graphs show the mean read density of H3K27Ac in NPCs and GPCs at group I and II

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

1]. (F) H3K27Ac read density in NPCs or GPCs around DNA-regions bound by SOX3 in NPCs. SOX3 DNA-regions with different H3K27Ac read density patterns in NPCs and GPCs were categorized as group I and II loci. Graphs show the mean read density of H3K27Ac in NPCs and GPCs at group I and II loci


( G ) Expression pattern of genes associated with group I and II loci ( from Fig . 2E ) within differentially expressed gene sets . Significance calculated by prop . test R , ( * * * ) P < 0 . 001 . ( H ) Venn diagram shows overlap between SOX3 binding in NPCs and GPCs . Bar graph shows expression pattern of genes continuously bound by SOX3 NPCs and GPCs . ( I ) Venn diagram shows overlap between SOX3 and SOX9 binding in GPCs . Bar graph shows expression pattern of genes co - bound by SOX3 and SOX9 in GPCs . ( J ) ChIP - seq peak graphics around the astrocyte gene Fgfbp3 . ChIP - seq peaks are derived from three different experiments ; SOX3 ChIPs in NPCs , SOX3 ChIPs in GPCs , SOX9 ChIPs in GPCs . Both ChIP - seq reads and called peak regions ( underlying black lines ) are shown for all data sets . Bar graphs shows the distribution of differentially expressed genes that are bound by all three factors . P - values ( phyper , R ) were calculated from the total number of protein coding genes in mm10 assembly ( 23 ´ 389 )

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

(G) Expression pattern of genes associated with group I and II loci (from Fig. 2E) within differentially expressed gene sets. Significance calculated by prop.test R, (***) P<0.001. (H) Venn diagram shows overlap between SOX3 binding in NPCs and GPCs. Bar graph shows expression pattern of genes continuously bound by SOX3 NPCs and GPCs. (I) Venn diagram shows overlap between SOX3 and SOX9 binding in GPCs. Bar graph shows expression pattern of genes co-bound by SOX3 and SOX9 in GPCs. (J) ChIP-seq peak graphics around the astrocyte gene Fgfbp3. ChIP-seq peaks are derived from three different experiments; SOX3 ChIPs in NPCs, SOX3 ChIPs in GPCs, SOX9 ChIPs in GPCs. Both ChIP-seq reads and called peak regions (underlying black lines) are shown for all data sets. Bar graphs shows the distribution of differentially expressed genes that are bound by all three factors. P-values (phyper, R) were calculated from the total number of protein coding genes in mm10 assembly (23´389)


( A ) Graphical view showing SOX9 and SOX10 expression among spinal cord cells analyzed with scRNA -

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

(A) Graphical view showing SOX9 and SOX10 expression among spinal cord cells analyzed with scRNA-seq


( B ) Bar graph shows enrichment of genes bound by SOX10 in rat oligodendrocytes within the differentially expressed gene sets . Yellow bar represents enrichment within NPC / GPC genes , green bar within neuronal genes , blue bar within astrocytic genes and red bar within oligodendrocytic genes . SOX10 ChIP - seq data from [

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

(B) Bar graph shows enrichment of genes bound by SOX10 in rat oligodendrocytes within the differentially expressed gene sets. Yellow bar represents enrichment within NPC/GPC genes, green bar within neuronal genes, blue bar within astrocytic genes and red bar within oligodendrocytic genes. SOX10 ChIP-seq data from [37


( F ) Expression of Nfia , Ehf and Zbtb3 among spinal cord cells analyzed with scRNA - seq . Red color indicates cells with RPKM values above 55 for Nfia , 5 for Zbtb3 and 5 for Ehf . n = 3 for SOX9 and 2 for SOX3 . P - values ( phyper , R ) were calculated from the total number of genes for mm10 ( 23 ´ 389 )

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

(F) Expression of Nfia, Ehf and Zbtb3 among spinal cord cells analyzed with scRNA-seq. Red color indicates cells with RPKM values above 55 for Nfia, 5 for Zbtb3 and 5 for Ehf. n=3 for SOX9 and 2 for SOX3. P-values (phyper, R) were calculated from the total number of genes for mm10 (23´389)


( A ) Luc - reporter assays in P19 cells . Luc - reporters containing SOX3 and SOX9 bound CRMs around the astrocyte specific genes Fgfbp3 and Ttyh1 were strongly activated by SOX9 in synergy with NFIA ; an activation that could be efficiently blocked by SOX3 . No activation of these reporters could be detected with SOX10 or SOX11 . ( B ) Luc - reporters containing SOX9 and SOX10 bound DNA - regions around the astrocyte genes Aldh1l1 and Atp1a2 , which are not expressed in SOX10 + oligodendrocytes , could not be activated by SOX9 or SOX10

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

(A) Luc-reporter assays in P19 cells. Luc-reporters containing SOX3 and SOX9 bound CRMs around the astrocyte specific genes Fgfbp3 and Ttyh1 were strongly activated by SOX9 in synergy with NFIA; an activation that could be efficiently blocked by SOX3. No activation of these reporters could be detected with SOX10 or SOX11. (B) Luc-reporters containing SOX9 and SOX10 bound DNA-regions around the astrocyte genes Aldh1l1 and Atp1a2, which are not expressed in SOX10+ oligodendrocytes, could not be activated by SOX9 or SOX10


( C ) Luc - reporters containing SOX9 and SOX10 bound CRMs around the oligodendrocyte specific genes Plekhb1 and Mid2 were activated in an additive manner by SOX9 and SOX10 . No activation could be detected with SOX11 . ( D ) Luc - reporters containing SOX3 and SOX11 bound CRMs of the neuronal genes Tubb3 and Lhx2 , which are sequentially bound by SOX3 and SOX11 , could be activated by SOX11 and efficiently repressed by SOX3 . No activation could be detected with

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

(C) Luc-reporters containing SOX9 and SOX10 bound CRMs around the oligodendrocyte specific genes Plekhb1 and Mid2 were activated in an additive manner by SOX9 and SOX10. No activation could be detected with SOX11. (D) Luc-reporters containing SOX3 and SOX11 bound CRMs of the neuronal genes Tubb3 and Lhx2, which are sequentially bound by SOX3 and SOX11, could be activated by SOX11 and efficiently repressed by SOX3. No activation could be detected with SOX10


( E ) Forced expression of SOX3 for 96 hrs in differentiating GPCs blocked the formation of astrocytes but not oligodendrocytes . Scale bar , 25µm . ( F ) Statistical view of Fig . 5E . Bars represents mean values ( of 3 experiments ) of cells expressing GFAP or SOX10 in GFP or Sox3 - transduced cells . Error bars in figure 5 indicate standard deviation of 3 - 7 individual samples and three - star significance > 0 . 001 ( t - test ) . ( * ) 0 , 05 < P < 0 , 01 ; ( * * ) 0 , 01 < P < 0 , 001 ; ( * * * ) P < 0 ,

O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Forced expression of SOX3 for 96 hrs in differentiating GPCs blocked the formation of astrocytes but not oligodendrocytes. Scale bar, 25µm. (F) Statistical view of Fig. 5E. Bars represents mean values (of 3 experiments) of cells expressing GFAP or SOX10 in GFP or Sox3-transduced cells. Error bars in figure 5 indicate standard deviation of 3-7 individual samples and three-star significance > 0.001 (t-test). (*) 0,05<P<0,01; (**) 0,01<P<0,001; (***) P<0,001


( A ) SOX3 over - expression experiments in chicken spinal cord at E4 . 48 hrs post electroporation , transfected side showed decreased expression of FGFR2 , NFIA and Fgfr3 . Bar : 50µm . ( B ) 24 hrs after miss - expression of a dominant negative version of SOXB1 ( dnSOXB1 ) , premature expression of FGFR2 , NFIA and Fgfr3 were found in the transfected side . Bar : 40µm . ( C ) 24 hrs after SOX9 over - expression in chicken spinal cord ( at E4 ) , increased expression of FGFR2 , NFIA and Fgfr3 was shown compared to the un - transfected control side . Bar : 40µm . ( D ) Sox3 and Sox9 co - electroporation in chicken spinal cord at E4 could rescue the effect of SOX9 overexpression and FGFR2 , NFIA and Fgfr3 levels were comparable to the un - transfected control side . Embryos were analyzed 24 hrs post electroporation . Bar : 40µm . ( E

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

(A) SOX3 over-expression experiments in chicken spinal cord at E4. 48 hrs post electroporation, transfected side showed decreased expression of FGFR2, NFIA and Fgfr3. Bar: 50µm. (B) 24 hrs after miss-expression of a dominant negative version of SOXB1 (dnSOXB1), premature expression of FGFR2, NFIA and Fgfr3 were found in the transfected side. Bar: 40µm. (C) 24 hrs after SOX9 over-expression in chicken spinal cord (at E4), increased expression of FGFR2, NFIA and Fgfr3 was shown compared to the un-transfected control side. Bar: 40µm. (D) Sox3 and Sox9 co-electroporation in chicken spinal cord at E4 could rescue the effect of SOX9 overexpression and FGFR2, NFIA and Fgfr3 levels were comparable to the un-transfected control side. Embryos were analyzed 24 hrs post electroporation. Bar: 40µm. (E


( B ) Representative images of cells released from mitosis for 1 , 2 or 3h in EdU - containing medium . Top , DNA staining with DAPI ; Bottom , EdU immunofluorescence . L , M and H indicate cells with low , medium or high levels of EdU incorporation . The barplot shows the quantification of the proportion of replicating cells at each time point .

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(B) Representative images of cells released from mitosis for 1, 2 or 3h in EdU-containing medium. Top, DNA staining with DAPI; Bottom, EdU immunofluorescence. L, M and H indicate cells with low, medium or high levels of EdU incorporation. The barplot shows the quantification of the proportion of replicating cells at each time point.


Y - axis , z - score ; X - axis , timepoints . A indicates asynchronous cells , M mitotic cells and the numbers represent the minutes after release from the mitotic block ) . Dots show individual replicates and lines averages ( n = 3 for A and M ; n = 2 for each post - mitotic sample ) . Pre - mRNA levels are shown for pluripotency genes ( Oct4 , Klf4 , Esrrb ) and cell cycle regulators ( E2f1 , Cdk2 , Rb ) ; mRNA levels for individual histone genes ( only averages are shown ) .

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

Y-axis, z-score; X-axis, timepoints. A indicates asynchronous cells, M mitotic cells and the numbers represent the minutes after release from the mitotic block). Dots show individual replicates and lines averages (n=3 for A and M; n=2 for each post-mitotic sample). Pre-mRNA levels are shown for pluripotency genes (Oct4, Klf4, Esrrb) and cell cycle regulators (E2f1, Cdk2, Rb); mRNA levels for individual histone genes (only averages are shown).


( C ) Percentage of genes presenting at least 1 TF binding site as detected by ChIP - seq within 25kb centred on the TSS , calculated for each gene category shown in ( A ) .

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

(C) Percentage of genes presenting at least 1 TF binding site as detected by ChIP-seq within 25kb centred on the TSS, calculated for each gene category shown in (A).


( B ) Heatmaps presented for untreated ( - IAA ; left ) or CTCF - depleted ( + IAA ; right ) CTCF - AID cells .

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

(B) Heatmaps presented for untreated (-IAA; left) or CTCF-depleted (+IAA; right) CTCF-AID cells.


( A ) Gene expression changes ( z - score ) in asynchronous cells and during post - mitotic release of CTCF - AID cells treated or not with IAA to deplete CTCF ( blue , presence of CTCF ; red , absence ) . Samples are indicated on the X - axis ( A : asynchronous cells ; M : mitotic cells ; numbers : minutes since release from mitotic block ) . Six gene groups are shown , based on the time of their response to CTCF depletion ( asy , late , early ) and the direction of the transcriptional change ( up , down ) . The number of genes of each group is indicated . The yellow shadow areas indicate times showing expression changes . The boxplots show the median ( bar ) , 25 - 75 % percentiles ( box ) and 1 . 5 - fold inter - quartile range ( whiskers ) .

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

(A) Gene expression changes (z-score) in asynchronous cells and during post-mitotic release of CTCF-AID cells treated or not with IAA to deplete CTCF (blue, presence of CTCF; red, absence). Samples are indicated on the X-axis (A: asynchronous cells; M: mitotic cells; numbers: minutes since release from mitotic block). Six gene groups are shown, based on the time of their response to CTCF depletion (asy, late, early) and the direction of the transcriptional change (up, down). The number of genes of each group is indicated. The yellow shadow areas indicate times showing expression changes. The boxplots show the median (bar), 25-75% percentiles (box) and 1.5-fold inter-quartile range (whiskers).


( C ) Average binding profile ( X - axis , reads per million ) of CTCF ( top ) and the Cohesin subunit SMC1 ( bottom ) , across 8kb centred on the TSS of genes responding to CTCF depletion exclusively in asynchronous cells ( left ) or either late or early upon mitotic release ( middle and right , respectively ) . Interphase is shown in blue and mitosis in red .

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

(C) Average binding profile (X-axis, reads per million) of CTCF (top) and the Cohesin subunit SMC1 (bottom), across 8kb centred on the TSS of genes responding to CTCF depletion exclusively in asynchronous cells (left) or either late or early upon mitotic release (middle and right, respectively). Interphase is shown in blue and mitosis in red.


B . Western blot of PTEN variants in a panel of human cancer cell lines using a rabbit monoclonal antibody against the C - terminal region of PTEN The PTEN null prostate cancer cell line PC3 was used as a negative control .

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

B. Western blot of PTEN variants in a panel of human cancer cell lines using a rabbit monoclonal antibody against the C-terminal region of PTEN The PTEN null prostate cancer cell line PC3 was used as a negative control.


D . Verification of expression of FLAG - tagged PTENα , PTENβ , and PTENε in PtenFLAG knock - in mice . Various tissues from PtenFLAG knock - in mice or control wild - type mice were lysed for immunoprecipitation with anti - FLAG M2 magnetic beads before being immunoblotted with the anti - PTENα

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

D. Verification of expression of FLAG-tagged PTENα, PTENβ, and PTENε in PtenFLAG knock-in mice. Various tissues from PtenFLAG knock-in mice or control wild-type mice were lysed for immunoprecipitation with anti-FLAG M2 magnetic beads before being immunoblotted with the anti-PTENα antibody


B . Reduction of PTENε in response to eIF2A knockdown . Hela cells were infected with lentivirus expressing eIF2A shRNA , eIF5 shRNA , or scramble shRNA separately . The knockdown of eIF2A or eIF5 was validated by western blot ( left panel ) . Lysates of Hela cells were collected and subjected to western blot analysis with antibodies against PTEN , and GAPDH ( right panel ) .

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B. Reduction of PTENε in response to eIF2A knockdown. Hela cells were infected with lentivirus expressing eIF2A shRNA, eIF5 shRNA, or scramble shRNA separately. The knockdown of eIF2A or eIF5 was validated by western blot (left panel). Lysates of Hela cells were collected and subjected to western blot analysis with antibodies against PTEN, and GAPDH (right panel).


C . Overexpression of eIF2A induces the relative expression of PTENε . FLAG - tagged eIF2A or eIF5 were overexpressed in DU145 cells before western blot analysis of PTENε expression with an antibody against PTEN . GAPDH was used as a loading control .

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

C. Overexpression of eIF2A induces the relative expression of PTENε. FLAG-tagged eIF2A or eIF5 were overexpressed in DU145 cells before western blot analysis of PTENε expression with an antibody against PTEN. GAPDH was used as a loading control.


C . The transfected Hela PTEN - / - cells were stained with an anti - β - catenin antibody and DAPI before being imaged with confocal microscopy .

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C. The transfected Hela PTEN-/- cells were stained with an anti-β-catenin antibody and DAPI before being imaged with confocal microscopy.


To determine the sequences that are critical for the membrane localization of PTENε . Hela were transfected with constructs Thirty - six hours after transfection , transfected Hela cells were stained with anti - β - catenin antibody and DAPI , followed by imaging with confocal microscopy . The scale bars represent 5 μm ( F ) .

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To determine the sequences that are critical for the membrane localization of PTENε. Hela were transfected with constructs Thirty-six hours after transfection, transfected Hela cells were stained with anti-β-catenin antibody and DAPI, followed by imaging with confocal microscopy. The scale bars represent 5 μm (F).


The transfected HEK293 cells were conducted with cell fractionation before western blotting with GFP , E - cadherin , and β - tubulin antibodies ( G ) . C : cytoplasm . M : cell plasma membrane .

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

The transfected HEK293 cells were conducted with cell fractionation before western blotting with GFP, E-cadherin, and β-tubulin antibodies (G). C: cytoplasm. M: cell plasma membrane.


D . Images of immunofluorescence staining for PTENε and its interacting proteins ( VASP and FSCN1 ) . Hela PTEN - / - cells were co - transfected by a plasmid expressing C - terminal GFP - tagged PTENε with FLAG - tagged targets ( FSCN1 , VASP ) separately , followed by staining with an anti - FLAG antibody and DAPI , and were imaged by confocal microscopy .

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

D. Images of immunofluorescence staining for PTENε and its interacting proteins (VASP and FSCN1). Hela PTEN-/- cells were co-transfected by a plasmid expressing C-terminal GFP-tagged PTENε with FLAG-tagged targets (FSCN1, VASP) separately, followed by staining with an anti-FLAG antibody and DAPI, and were imaged by confocal microscopy.


E . The cell plasma membrane localization of PTENε is required for its interaction with downstream targets . FLAG - tagged CDC42 , VASP , ACTR2 , or FSCN1 plasmid was co - transfected with a plasmid expressing PTENε with or without 69 - 81aa deletion carrying C - terminal S - tag and HA tag in HEK293 cells . Cell lysates were incubated with S protein agarose before western blotting with antibodies against the FLAG tag and HA tag .

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

E. The cell plasma membrane localization of PTENε is required for its interaction with downstream targets. FLAG-tagged CDC42, VASP, ACTR2, or FSCN1 plasmid was co-transfected with a plasmid expressing PTENε with or without 69-81aa deletion carrying C-terminal S-tag and HA tag in HEK293 cells. Cell lysates were incubated with S protein agarose before western blotting with antibodies against the FLAG tag and HA tag.


( H , I ) Purified ACTR2 or VASP was phosphorylated in vitro by NIK or PKA and subsequently used in a phosphatase assay with His - PTENε or without purified PTENε protein as a control , followed by immunoblotting with antibodies against p - ACTR2 ( Thr237 and Thr238 ) and p - VASP ( Ser157 ) . Purified proteins ( PTENε , PKA , NIK , ACTR , and VASP ) were detected by anti - His antibody ( ZSGB - BIO , TA - 02 ) .

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(H, I) Purified ACTR2 or VASP was phosphorylated in vitro by NIK or PKA and subsequently used in a phosphatase assay with His-PTENε or without purified PTENε protein as a control, followed by immunoblotting with antibodies against p-ACTR2 (Thr237 and Thr238) and p-VASP (Ser157). Purified proteins (PTENε, PKA, NIK, ACTR, and VASP) were detected by anti-His antibody (ZSGB-BIO, TA-02).


A . Immunofluorescence staining of F - actin in PTENε or PTENε Y210L overexpressed Hela PTEN - / - cells and control cells . PTENε - GFP , PTENε Y210L - GFP , or mock construct was introduced into HeLa PTEN - / - cells respectively . The transfected cells were stained with Phalloidin and DAPI before being imaged with confocal microscopy . The scale bars represent 5 μm ( left panel ) . White arrows shown in the magnified immunofluorescence images indicate the filopodia in the cell membrane . B . Quantification of the number of filopodia per cell by FiloQuant software and the data are presented as mean ± SD of three independent experiments and were analyzed with the unpaired t - test . * * * * , p < 0 . 001 .

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

A. Immunofluorescence staining of F-actin in PTENε or PTENε Y210L overexpressed Hela PTEN-/- cells and control cells. PTENε-GFP, PTENε Y210L-GFP, or mock construct was introduced into HeLa PTEN-/- cells respectively. The transfected cells were stained with Phalloidin and DAPI before being imaged with confocal microscopy. The scale bars represent 5 μm (left panel). White arrows shown in the magnified immunofluorescence images indicate the filopodia in the cell membrane. B. Quantification of the number of filopodia per cell by FiloQuant software and the data are presented as mean ± SD of three independent experiments and were analyzed with the unpaired t-test. ****, p<0.001.


G . Left panel : immunoblotting analyses were used to verify the level of overexpressed PTENε or PTENε Y210L in B16 cells . Middle panel : representative images showing the lung metastasis of PTENε or PTENε Y210L overexpressed B16 cells or control cells in the mouse pulmonary metastasis model ( n = 4 ) . H & E , hematoxylin - eosin staining . The scale bars represent 600 mm ( upper panel ) and 500μm ( lower panel ) separately . Right panel : the representative bar graphs showing the quantification of tumor metastatic ability was displayed by measuring the number of tumor nodules ( data are presented as mean ± SD based on three independent replicates ) . The overexpression of PTENε attenuated tumor metastatic ability compared with the vector control group . * * * * , p < 0 . 0001 in the unpaired t - test .

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

G. Left panel: immunoblotting analyses were used to verify the level of overexpressed PTENε or PTENε Y210L in B16 cells. Middle panel: representative images showing the lung metastasis of PTENε or PTENε Y210L overexpressed B16 cells or control cells in the mouse pulmonary metastasis model (n=4). H & E, hematoxylin-eosin staining. The scale bars represent 600 mm (upper panel) and 500μm (lower panel) separately. Right panel: the representative bar graphs showing the quantification of tumor metastatic ability was displayed by measuring the number of tumor nodules (data are presented as mean ± SD based on three independent replicates). The overexpression of PTENε attenuated tumor metastatic ability compared with the vector control group. ****, p<0.0001 in the unpaired t-test.


( A and B ) THP1 - derived macrophages were treated with the TLR ligands Pam3 , lipopolysaccharide ( LPS ) , or FSL - 1 and analyzed by RNA - seq 8 h later . Circos plots show genome - wide differential expression of protein - coding genes ( A ) and non - protein - coding genes ( B ) between untreated and treated macrophages . The outermost to innermost circles show downregulated ( blue ) or upregulated ( red ) genes in cells stimulated with FSL - 1 , LPS , and Pam3 , respectively . Chromosomes are indicated by the numbers 1 - 22 and X and Y .

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

(A and B) THP1-derived macrophages were treated with the TLR ligands Pam3, lipopolysaccharide (LPS), or FSL-1 and analyzed by RNA-seq 8 h later. Circos plots show genome-wide differential expression of protein-coding genes (A) and non-protein-coding genes (B) between untreated and treated macrophages. The outermost to innermost circles show downregulated (blue) or upregulated (red) genes in cells stimulated with FSL-1, LPS, and Pam3, respectively. Chromosomes are indicated by the numbers 1-22 and X and Y.


( F ) RT - qPCR validation of differentially expressed lncRNAs and mRNAs in THP1 - derived macrophages stimulated for 8 h with flagellin ( FLA ) , FSL - 1 , imiquimod , LPS , Pam3 , or poly ( I : C ) . Heat map shows the fold change in expression of 13 mRNAs ( left ) and lncRNAs ( right ) .

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(F) RT-qPCR validation of differentially expressed lncRNAs and mRNAs in THP1-derived macrophages stimulated for 8 h with flagellin (FLA), FSL-1, imiquimod, LPS, Pam3, or poly(I:C). Heat map shows the fold change in expression of 13 mRNAs (left) and lncRNAs (right).


( A ) Activation and suppression of nearby genes by eight cis - acting lncRNAs . RT - qPCR analysis of THP1 - derived macrophages transduced with lentiviruses expressing non - targeting control ( NC ) shRNA or shRNAs targeted to the indicated lncRNAs and protein - coding genes . Mean ± SD of n = 3 . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 0001 by Student ' s t test .

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(A) Activation and suppression of nearby genes by eight cis-acting lncRNAs. RT-qPCR analysis of THP1-derived macrophages transduced with lentiviruses expressing non-targeting control (NC) shRNA or shRNAs targeted to the indicated lncRNAs and protein-coding genes. Mean ± SD of n = 3. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001 by Student's t test.


Silencing of cis - regulated gene pairs affects GBS - induced expression of IL - 6 RT - qPCR analysis of THP1 - derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h , infected with GBS for 1 h , and then treated with gentamicin for 30 min to terminate infection . RT - qPCR were performed at 10 h post - infection .

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Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-6 RT-qPCR analysis of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. RT-qPCR were performed at 10 h post-infection.


Silencing of cis - regulated gene pairs affects GBS - induced expression of IL - 6 IL - 6 ELISA quantification ( C ) of THP1 - derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h , infected with GBS for 1 h , and then treated with gentamicin for 30 min to terminate infection . ELISA were performed at 10 h post - infection .

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Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-6 IL-6 ELISA quantification (C) of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. ELISA were performed at 10 h post-infection.


Silencing of cis - regulated gene pairs affects GBS - induced expression of IL - 1α ( D ) . RT - qPCR analysis of THP1 - derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h , infected with GBS for 1 h , and then treated with gentamicin for 30 min to terminate infection . RT - qPCR were performed at 10 h post - infection .

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Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-1α (D). RT-qPCR analysis of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. RT-qPCR were performed at 10 h post-infection.


lnc - MARCKS ( B ) expressions were quantified in cells stably expressing dCas9 / KRAB / BFP and the NC or sgRNAs .

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lnc-MARCKS (B) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs.


, MARCKS ( C ) expressions were quantified in cells stably expressing dCas9 / KRAB / BFP and the NC or sgRNAs .

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, MARCKS (C) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs.


IL - 6 ( D ) expressions were quantified in cells stably expressing dCas9 / KRAB / BFP and the NC or sgRNAs .

IL-6 (D) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs.


Overexpression of lnc - MARCKS decreases MARCKS expression RT - qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector ( E . V . ) or lnc - MARCKS , treated with PMA overnight , and left unstimulated or stimulated with Pam3 for 8 h . Lnc - MARCKS and MARCKS expression ( F )

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Overexpression of lnc-MARCKS decreases MARCKS expression RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. Lnc-MARCKS and MARCKS expression (F)


Overexpression of lnc - MARCKS enhances IL - 6 expression . RT - qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector ( E . V . ) or lnc - MARCKS , treated with PMA overnight , and left unstimulated or stimulated with Pam3 for 8 h . IL - 6 expression ( G ) .

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Overexpression of lnc-MARCKS enhances IL-6 expression. RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. IL-6 expression (G).


lnc - MARCKS regulates MARCKS in primary macrophages . Primary macrophages were transfected with NC or lnc - MARCKS antisense oligo to knock down lnc - MARCKS expression . 48 h later , cells were stimulated with Pam3 for 8 h and harvested to detect lnc - MARCKS ( H )

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lnc-MARCKS regulates MARCKS in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect lnc-MARCKS (H)


lnc - MARCKS regulates MARCKS in primary macrophages . Primary macrophages were transfected with NC or lnc - MARCKS antisense oligo to knock down lnc - MARCKS expression . 48 h later , cells were stimulated with Pam3 for 8 h and harvested to detect MARCKS ( I ) ,

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lnc-MARCKS regulates MARCKS in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect MARCKS (I),


lnc - MARCKS regulates IL - 6 in primary macrophages . Primary macrophages were transfected with NC or lnc - MARCKS antisense oligo to knock down lnc - MARCKS expression . 48 h later , cells were stimulated with Pam3 for 8 h and harvested to detect IL - 6 expressions ( J ) .

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lnc-MARCKS regulates IL-6 in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect IL-6 expressions (J).


( A ) ROCKI is recruited to the MARCKS promoter in Pam3 - stimulated THP1 - derived macrophages . ChIRP analysis of THP1 - derived macrophages treated with Pam3 for 8 h . ChIRP assays were performed using a non - specific lacZ probe or probes specific for lnc - MARCKS . Specificity of ROCKI probes ( left ) , and enrichment at the MARCKS promoter ( right ) .

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(A) ROCKI is recruited to the MARCKS promoter in Pam3-stimulated THP1-derived macrophages. ChIRP analysis of THP1-derived macrophages treated with Pam3 for 8 h. ChIRP assays were performed using a non-specific lacZ probe or probes specific for lnc-MARCKS. Specificity of ROCKI probes (left), and enrichment at the MARCKS promoter (right).


( B ) Mass spectrometry identifies APEX1 as a candidate ROCKI - binding protein in THP1 - derived macrophages . APEX1 MS / MS count represents the ratio between the number of APEX1 peptides and all peptides detected . Mean of n = 2 samples . ROCKI and ROCKI - AS represent ROCKI and antisense strand of ROCKI transcripts , respectively .

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(B) Mass spectrometry identifies APEX1 as a candidate ROCKI-binding protein in THP1-derived macrophages. APEX1 MS/MS count represents the ratio between the number of APEX1 peptides and all peptides detected. Mean of n = 2 samples. ROCKI and ROCKI-AS represent ROCKI and antisense strand of ROCKI transcripts, respectively.


( C ) ROCKI inhibition of MARCKS expression is dependent on APEX1 . RT - qPCR analysis of MARCKS , ROCKI , and APEX1 expression in THP1 - derived macrophages overexpressing ROCKI and / or depleted of APEX1 .

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(C) ROCKI inhibition of MARCKS expression is dependent on APEX1. RT-qPCR analysis of MARCKS, ROCKI, and APEX1 expression in THP1-derived macrophages overexpressing ROCKI and/or depleted of APEX1.


( D ) APEX1 specially binds to ROCKI . Western blot analysis of APEX1 in lacZ , or ROCKI pull - down fractions . Vimentin , which non - specifically binds to all RNA probes , is shown as a loading control .

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(D) APEX1 specially binds to ROCKI. Western blot analysis of APEX1 in lacZ, or ROCKI pull-down fractions. Vimentin, which non-specifically binds to all RNA probes, is shown as a loading control.


( E ) ROCKI associates with APEX1 . Left : RT - qPCR analysis of ROCKI present in anti - APEX1 antibody or control IgG immunoprecipitates from nuclear lysates of Pam3 - treated THP1 - derived macrophages . Right : APEX1 western blot of the same immunoprecipitates .

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(E) ROCKI associates with APEX1. Left: RT-qPCR analysis of ROCKI present in anti-APEX1 antibody or control IgG immunoprecipitates from nuclear lysates of Pam3-treated THP1-derived macrophages. Right: APEX1 western blot of the same immunoprecipitates.


ChIP analysis of THP1 - derived macrophages demonstrates APEX1 recruitment to the MARCKS promoter . RT - qPCR analysis of fragments of the MARCKS promoter region ( a and b ) or a non - specific region ( nc ) co - immunoprecipitated with anti - APEX1 antibody .

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ChIP analysis of THP1-derived macrophages demonstrates APEX1 recruitment to the MARCKS promoter. RT-qPCR analysis of fragments of the MARCKS promoter region (a and b) or a non-specific region (nc) co-immunoprecipitated with anti-APEX1 antibody.


ChIP analysis of APEX1 recruitment to the MARCKS promoter was performed except the cells expressed control shRNA ( NC ) or ROCKI shRNA and treated with Pam3 for 8 h . The y - axis shows the percentage of enrichment in normalized to input . An unrelated region of the ACTB2 promoter was amplified as a control .

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ChIP analysis of APEX1 recruitment to the MARCKS promoter was performed except the cells expressed control shRNA (NC) or ROCKI shRNA and treated with Pam3 for 8 h. The y-axis shows the percentage of enrichment in normalized to input. An unrelated region of the ACTB2 promoter was amplified as a control.


Left : Western blot analysis of APEX1 and HDAC1 in control rabbit IgG or anti - APEX1 antibody immunoprecipitates from NC ( control ) or ROCKI knockdown THP1 - derived macrophages . Right : RT - qPCR analysis of ROCKI in the same immunoprecipitates .

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Left: Western blot analysis of APEX1 and HDAC1 in control rabbit IgG or anti-APEX1 antibody immunoprecipitates from NC (control) or ROCKI knockdown THP1-derived macrophages. Right: RT-qPCR analysis of ROCKI in the same immunoprecipitates.


Knockdown of ROCKI decreased HDAC1 enrichment on the MARCKS promoter . ChIP - qPCR analysis of HDAC1 was performed using an anti - HDAC1 antibody .

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Knockdown of ROCKI decreased HDAC1 enrichment on the MARCKS promoter. ChIP-qPCR analysis of HDAC1 was performed using an anti- HDAC1 antibody.


Knockdown of ROCKI enhances H3K27ac deposition on the MARCKS promoter . ChIP - qPCR analysis of H3K27ac was performed using an anti - H3K27ac antibody .

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Knockdown of ROCKI enhances H3K27ac deposition on the MARCKS promoter. ChIP-qPCR analysis of H3K27ac was performed using an anti-H3K27ac antibody.


Heatmap of representative genes commonly upregulated in ROCKI - overexpressing and MARCKS - knockdown THP1 - derived macrophages .

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Heatmap of representative genes commonly upregulated in ROCKI-overexpressing and MARCKS-knockdown THP1-derived macrophages.


RT - qPCR validation of inflammatory gene expression in ROCKI - overexpressing and MARCKS - knockdown THP1 - derived macrophages stimulated with Pam3 for 8 h . E . V . , empty vector ; NC , control shRNA .

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RT-qPCR validation of inflammatory gene expression in ROCKI-overexpressing and MARCKS-knockdown THP1-derived macrophages stimulated with Pam3 for 8 h. E.V., empty vector; NC, control shRNA.


MARCKS is a negative regulator of Ca2 + signaling . Fluo - 4 fluorescence of MARCKS , ROCKI , or NC ( control ) shRNA - expressing THP1 - derived macrophages before and after treatment with PMA and Pam3 . Triton X - 100 was added to obtain the maximal [ Ca2 + ] signal . Fluorescence levels are expressed as the change in signal normalized to the maximal fluorescence ( ΔF / Fmax ) .

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MARCKS is a negative regulator of Ca2+ signaling. Fluo-4 fluorescence of MARCKS, ROCKI, or NC (control) shRNA-expressing THP1-derived macrophages before and after treatment with PMA and Pam3. Triton X-100 was added to obtain the maximal [Ca2+] signal. Fluorescence levels are expressed as the change in signal normalized to the maximal fluorescence (ΔF/Fmax).


( A ) QTL signals for ROCKI ( LINC01268 ) gene expression in whole blood ( top , black ) , H3K27ac promoter signal in monocytes ( middle , green ) , and H3K4me1 signal in monocytes ( bottom , orange ) in a 400 kb window around the MARCKS / ROCKI locus . The signals were strongly co - localized ( Pshared = . 97 , Pshared = . 98 ) suggesting they represent the same signal .

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(A) QTL signals for ROCKI (LINC01268) gene expression in whole blood (top, black), H3K27ac promoter signal in monocytes (middle, green), and H3K4me1 signal in monocytes (bottom, orange) in a 400 kb window around the MARCKS/ROCKI locus. The signals were strongly co-localized (Pshared=.97, Pshared=.98) suggesting they represent the same signal.


( C ) RT - qPCR analysis of ROCKI expression in dendritic cell samples from uninfected and M . tuberculosis - infected donors .

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(C) RT-qPCR analysis of ROCKI expression in dendritic cell samples from uninfected and M. tuberculosis-infected donors.


D , Affinity capture of proteins by OC43p and OC43 - SCR bound magnetic beads . Specific reactivity to Atp1a1 and Atp1a2 was detected by protein immunoblotting using specific primary antibodies . OC43p - SCR - coated and uncoated beads ( Beads ) were used to control non - specific binding . Spinal cords were isolated from male ( M ) and female ( F ) rats ( n = 2 / group ) . Input protein lysates ( right side , in duplicates ) served as loading controls . Approximate molecular weights ( in kDa ) are indicated by arrows .

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D, Affinity capture of proteins by OC43p and OC43-SCR bound magnetic beads. Specific reactivity to Atp1a1 and Atp1a2 was detected by protein immunoblotting using specific primary antibodies. OC43p-SCR-coated and uncoated beads (Beads) were used to control non-specific binding. Spinal cords were isolated from male (M) and female (F) rats (n=2/group). Input protein lysates (right side, in duplicates) served as loading controls. Approximate molecular weights (in kDa) are indicated by arrows.


Determination of TAPBPL protein expression in normal and tumor human tissues by immunohistochemistry using anti - TAPBPL or isotype Ab .

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Determination of TAPBPL protein expression in normal and tumor human tissues by immunohistochemistry using anti-TAPBPL or isotype Ab.


Splenocytes from C57BL / 6 mice were freshly harvested . Resting and activated T cells , monocytes , macrophages , DCs and B cells were obtained as in Figure 2 . The resting and activated immune cells were stained with biotinylated TAPBPL - Ig or control Ig ， followed by streptavidin PE , as well as anti - CD4 , CD8 , CD11b , F4 / 80 , CD11c , B220 or CD19 antibody to identify immune cells . statistical analysis showing the binding TAPBPL - Ig or control Ig to freshly harvest and activated immune cells ( n = 3 ) . Significance was calculated by two ‐ way ANOVA with Tukey test . * P < 0 . 05 compared with control Ig . * * P < 0 . 05 compared with resting cells .

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Splenocytes from C57BL/6 mice were freshly harvested. Resting and activated T cells, monocytes, macrophages, DCs and B cells were obtained as in Figure 2. The resting and activated immune cells were stained with biotinylated TAPBPL-Ig or control Ig，followed by streptavidin PE, as well as anti-CD4, CD8, CD11b, F4/80, CD11c, B220 or CD19 antibody to identify immune cells. statistical analysis showing the binding TAPBPL-Ig or control Ig to freshly harvest and activated immune cells (n=3). Significance was calculated by two‐way ANOVA with Tukey test. * P<0.05 compared with control Ig. ** P<0.05 compared with resting cells.


Splenic cells from C57BL / 6 mice were cultured with anti - CD3 antibody and anti - CD28 ( 0 . 5 µg / ml ) antibodies in the presence of graded doses of hTAPBPL - Ig or equimolar amounts of control Ig ( 3 . 75 and 5 . 63 µg / ml ) for 1 day The cells were analyzed for CD69 + , cells in CD4 and CD8 T cells . Representative flow cytometric and statistical analyses of the percentages of CD69 + , and CD44loCD62Lhi Naïve T cells in CD4 and CD8 T cells . The data are expressed as mean + SD ( n = 3 ) . Significance was calculated by two ‐ tailed Student ' s t ‐ test . * P < 0 . 05 compared with control Ig .

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

Splenic cells from C57BL/6 mice were cultured with anti-CD3 antibody and anti-CD28 (0.5 µg/ml) antibodies in the presence of graded doses of hTAPBPL-Ig or equimolar amounts of control Ig (3.75 and 5.63 µg/ml) for 1 day The cells were analyzed for CD69+, cells in CD4 and CD8 T cells. Representative flow cytometric and statistical analyses of the percentages of CD69+, and CD44loCD62Lhi Naïve T cells in CD4 and CD8 T cells. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig.


Splenic cells were labelled with CFSE and cultured in 96 - well plates that were precoated with anti - CD3 antibody and hTAPBPL - Ig or control Ig for 3 days as in ( A ) . The cells were analyzed for CFSE levels by CD4 + and CD8 + T cells . statistical analysis of ( D ) CD4 and ( E ) CD8 T cell proliferation .

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

Splenic cells were labelled with CFSE and cultured in 96-well plates that were precoated with anti-CD3 antibody and hTAPBPL-Ig or control Ig for 3 days as in (A). The cells were analyzed for CFSE levels by CD4+ and CD8+ T cells. statistical analysis of (D) CD4 and (E) CD8 T cell proliferation.


Purified human T cells were cultured with plate - bound anti - human CD3 antibody ( 1 µg / ml ) in the presence of graded doses of hTAPBPL - Ig ( 1 . 5 and 3 µg / ml ) or control Ig protein ( 1 . 5 and 3 µg / ml ) for 3 days . Cell proliferation was measured by [ 3H ] thymidine incorporation . The data are expressed as mean + SD ( n = 3 ) . Significance was calculated by two ‐ tailed Student ' s t ‐ test . * P < 0 . 05 compared with control Ig .

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

Purified human T cells were cultured with plate-bound anti-human CD3 antibody (1 µg/ml) in the presence of graded doses of hTAPBPL-Ig (1.5 and 3 µg/ml) or control Ig protein (1.5 and 3 µg/ml) for 3 days. Cell proliferation was measured by [3H] thymidine incorporation. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig.


Anti - hTAPBPL mAb ( clone 54 ) neutralized the inhibitory activity of hTAPBPL - Ig on T cell proliferation Significance in was calculated by one ‐ way ANOVA with Dunnett test and others by two ‐ tailed Student ' s t ‐ test . * P < 0 . 05 , anti - hTAPBPL Ab group was compared with isotype Ab group .

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

Anti-hTAPBPL mAb (clone 54) neutralized the inhibitory activity of hTAPBPL-Ig on T cell proliferation Significance in was calculated by one‐way ANOVA with Dunnett test and others by two‐tailed Student's t‐test. * P<0.05, anti-hTAPBPL Ab group was compared with isotype Ab group.


DBA / 2J mice were injected s . c . with 1 × 105 P388 murine leukemia cells , followed by injection of the anti - hTAPBPL mAb ( 25 , 50 , or 100µg ) or isotype Ab ( 25 , 50 , or 100 µg ) 3 times per week . Tumors size was measured over time . The mean tumor diameter ( mm3 ) + S . D . at the indicated time points are shown .

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

DBA/2J mice were injected s.c. with 1× 105 P388 murine leukemia cells, followed by injection of the anti-hTAPBPL mAb (25, 50, or 100µg) or isotype Ab (25, 50, or 100 µg) 3 times per week. Tumors size was measured over time. The mean tumor diameter (mm3) + S.D. at the indicated time points are shown.


Tumor infiltrating CD4 + and CD8 + T cells were examined by immunofluorescence .

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

Tumor infiltrating CD4+ and CD8+ T cells were examined by immunofluorescence.


LDH assay was performed after 24 h of cold stress on ice . Data are presented as mean ± SEM ; n = 3 , biological replicates . * * * * p < 0 . 0001 , * * * p < 0 . 001 by one - way ANOVA followed by Dunnett ' s multiple comparison test .

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

LDH assay was performed after 24 h of cold stress on ice. Data are presented as mean ± SEM; n = 3, biological replicates. ****p < 0.0001, ***p < 0.001 by one-way ANOVA followed by Dunnett's multiple comparison test.


Immunoblots of MICU1 and its mutants rescued by the lentivirus . Arrows indicate MICU1 or the dimer . * indicates the potential DTT - resistant dimer .

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

Immunoblots of MICU1 and its mutants rescued by the lentivirus. Arrows indicate MICU1 or the dimer. * indicates the potential DTT-resistant dimer.


MMP was monitored by TMRE fluorescence at every 1 h . FCCP ( 200 µM ) and antimycin A ( 50 µM ) were used as pretreatments for 30 min . Data were normalized by basal state of WT A549 cells ( B - D ) Data at 1 h ( C ) , 5 h ( D are presented as mean ± SEM ; n = 4 ( B - D ) biological replicates , * * * * p < 0 . 0001 , * * p < 0 . 01 , * p < 0 . 05 by paired T - test ( C ) , or one - way ANOVA followed by Dunnett ' s multiple comparison test (

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

MMP was monitored by TMRE fluorescence at every 1 h. FCCP (200 µM) and antimycin A (50 µM) were used as pretreatments for 30 min. Data were normalized by basal state of WT A549 cells (B-D) Data at 1 h (C), 5 h (D are presented as mean ± SEM; n = 4 (B-D) biological replicates, ****p < 0.0001, **p < 0.01, *p < 0.05 by paired T-test (C), or one-way ANOVA followed by Dunnett's multiple comparison test (D