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

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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-MEASURED_VAR O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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


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

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


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

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


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

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

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


A . RT - qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human brown adipocytes in response to chronic treatments ( 8 hours ) with DMSO ( control ) , MP - 10 ( 1nM and 100nM ) , cyclic AMP ( cAMP - 10µM ) , cyclic GMP ( cGMP - 10µM ) and cAMP + cGMP ( cA + cG - 10µM ( n = 4 separate cultures ) . * P = 0 . 0278 vs ( 100nM MP - 10 vs control ; Pgc1alpha ) , * * P = 0 . 0097 ( 10µM cA + cG vs control ; Pgc1alpha ) , * * P = 0 . 0063 ( 1nM MP - 10 vs control ; Ucp1 ) , * * * * P < 0 . 0001 ( 100nM MP - 10 vs control ; Ucp1 ) , # # P = 0 . 0017 ( 1nM MP - 10 vs 100nM MP - 10 ; Ucp1 ) , § § § § P < 0 . 0001 ( 10µM cAMP vs 100nM MP - 10 ; Ucp1 ) , § § § § P < 0 . 0001 ( 10µM cGMP vs 100nM MP - 10 ; Ucp1 ) , § § § § P < 0 . 0001 ( 10µM cA + cG vs 100nM MP - 10 ; Ucp1 ) , * P = 0 . 0112 vs ( 100nM MP - 10 vs control ; Cidea ) , * * * P = 0 . 0007 ( 1nM MP - 10 vs control ; Dio2 ) , * * * * P < 0 . 0001 ( 100nM MP - 10 vs control ; Dio2 ) , # P = 0 . 0267 ( 1nM MP - 10 vs 10µM cAMP ; Dio2 ) , § § § P = 0 . 0001 ( 10µM cAMP vs 100nM MP - 10 ; Dio2 ) , § § § P = 0 . 0007 ( 10µM cGMP vs 100nM MP - 10 ; Dio2 ) , * P = 0 . 0232 ( 10µM cA + cG vs control ; Dio2 ) § § P = 0 . 0058 ( 10µM cA + cG vs 100nM MP - 10 ; Dio2 ) , * P = 0 . 0416 ( 10µM cGMP vs control ; Prdm16 ) and * P = 0 . 0105 ( 10µM cA + cG vs control ; Prdm16 ) using one - way analysis of variance ( ANOVA ) with Tukey ' s post - hoc test . B . RT - qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human white adipocytes in response to chronic treatments ( 8 hours ) with DMSO ( control ) , MP - 10 ( 1nM and 100nM ) , cyclic AMP ( cAMP - 10µM ) , cyclic GMP ( cGMP - 10µM ) and cAMP + cGMP ( cA + cG - 10µM ( n = 4 separate cultures ) . * P = 0 . 0186 vs ( 1nM MP - 10 vs control ; Ucp1 ) , * * * * P < 0 . 0001 ( 100nM MP - 10 vs control ; Ucp1 ) , # # P = 0 . 0016 ( 1nM MP - 10 vs 100nM MP - 10 ; Ucp1 ) , * * * P = 0 . 0003 ( 10µM cAMP vs control ; Ucp1 ) , § § § P = 0 . 0002 ( 10µM cGMP vs 100nM MP - 10 ; Ucp1 ) and * * * P = 0 . 0003 ( cA + cG vs control ; Ucp1 ) using one - way analysis of variance ( ANOVA ) with Tukey ' s post - hoc test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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. RT-qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human brown adipocytes in response to chronic treatments (8 hours) with DMSO (control), MP-10 (1nM and 100nM), cyclic AMP (cAMP - 10µM), cyclic GMP (cGMP - 10µM) and cAMP + cGMP (cA + cG - 10µM (n = 4 separate cultures). * P = 0.0278 vs (100nM MP-10 vs control; Pgc1alpha), **P = 0.0097 (10µM cA + cG vs control; Pgc1alpha), ** P = 0.0063 (1nM MP-10 vs control; Ucp1), **** P < 0.0001 (100nM MP-10 vs control; Ucp1), ## P = 0.0017 (1nM MP-10 vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cAMP vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cGMP vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cA + cG vs 100nM MP-10; Ucp1), * P = 0.0112 vs (100nM MP-10 vs control; Cidea), *** P = 0.0007 (1nM MP-10 vs control; Dio2), **** P < 0.0001 (100nM MP-10 vs control; Dio2), # P = 0.0267 (1nM MP-10 vs 10µM cAMP; Dio2), §§§ P = 0.0001 (10µM cAMP vs 100nM MP-10; Dio2), §§§ P = 0.0007 (10µM cGMP vs 100nM MP-10; Dio2), * P = 0.0232 (10µM cA + cG vs control; Dio2) §§ P = 0.0058 (10µM cA + cG vs 100nM MP-10; Dio2), * P = 0.0416 (10µM cGMP vs control; Prdm16) and * P = 0.0105 (10µM cA + cG vs control; Prdm16) using one-way analysis of variance (ANOVA) with Tukey's post-hoc test.B. RT-qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human white adipocytes in response to chronic treatments (8 hours) with DMSO (control), MP-10 (1nM and 100nM), cyclic AMP (cAMP - 10µM), cyclic GMP (cGMP - 10µM) and cAMP + cGMP (cA + cG - 10µM (n = 4 separate cultures). * P = 0.0186 vs (1nM MP-10 vs control; Ucp1), **** P < 0.0001 (100nM MP-10 vs control; Ucp1), ## P = 0.0016 (1nM MP-10 vs 100nM MP-10; Ucp1), *** P = 0.0003 (10µM cAMP vs control; Ucp1), §§§ P = 0.0002 (10µM cGMP vs 100nM MP-10; Ucp1) and *** P = 0.0003 (cA + cG vs control; Ucp1) using one-way analysis of variance (ANOVA) with Tukey's post-hoc test.


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( A ) Percentage body weight loss Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M

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Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (A) Percentage body weight loss Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( B ) Relative amount of PR8 viral NP mRNA , by qPCR Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M

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Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (B) Relative amount of PR8 viral NP mRNA, by qPCR Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( C ) Histology of lungs by H & E staining with pathology scores . Scale bars = 200 μm Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M

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Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (C) Histology of lungs by H&E staining with pathology scores. Scale bars = 200 μm Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( D ) Absolute number of live CD45 + cells in whole lungs and BAL , by flow cytometry Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M Statistical significanc was determined with unpaired student ' s T -

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Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (D) Absolute number of live CD45+ cells in whole lungs and BAL, by flow cytometry Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M Statistical significanc was determined with unpaired student's T-test


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( E ) Level of LDH in BAL , by LDH cytotoxicity assay Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (E) Level of LDH in BAL, by LDH cytotoxicity assay Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( F ) Expression of PDPN in lungs , by western blot Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M

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Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (F) Expression of PDPN in lungs, by western blot Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( G ) Total protein in BAL , by Bradford assay . ( H ) Concentration of albumin in BAL , by ELISA Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M . Statistical significanc was determined with unpaired student ' s T -

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Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (G) Total protein in BAL, by Bradford assay. (H) Concentration of albumin in BAL, by ELISA Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M. Statistical significanc was determined with unpaired student's T-test


( A ) Absolute cell counts from lungs of sub - lethally influenza virus infected mice following analysis by flow cytometry . After gating out alveolar macrophages by forward and side scatter , immune cells are defined as follows : Neutrophil ( Ly6G + CD11b + SiglecF - Ly6Cint ) , Eosinophil ( SiglecF + CD11c - / + F4 / 80 - CD64 - ) , Monocyte ( Ly6G - CD11b + MHCII - SiglecF - ) , Natural killer cell ( NK , B220 - / intNK1 . 1 + ) , B cell ( B220hiNK1 . 1 - ) , CD8 + T cell ( TCRβ + CD8 + ) , CD4 + T cell ( TCRβ + CD4 + ) Data information : Data are representative of three independent experiments with 3 - 4 mice per group and represented as mean ± S . E . M . Statistical significance in ( A ) was determined with unpaired student ' s T - test . n . s . = not significant

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(A) Absolute cell counts from lungs of sub-lethally influenza virus infected mice following analysis by flow cytometry. After gating out alveolar macrophages by forward and side scatter, immune cells are defined as follows: Neutrophil (Ly6G+CD11b+SiglecF-Ly6Cint), Eosinophil (SiglecF+CD11c-/+F4/80-CD64-), Monocyte (Ly6G-CD11b+MHCII-SiglecF-), Natural killer cell (NK, B220-/intNK1.1+), B cell (B220hiNK1.1-), CD8+ T cell (TCRβ+CD8+), CD4+ T cell (TCRβ+CD4+) Data information: Data are representative of three independent experiments with 3-4 mice per group and represented as mean ± S.E.M. Statistical significance in (A) was determined with unpaired student's T-test. n.s.= not significant


( B ) Flow cytometry plots of neutrophils and eosinophils in lungs at days 9 and 15 post sub - lethal influenza virus infection . Numbers in plots indicate frequency of gated immune cells in each throughput Data information : Data are representative of three independent experiments with 3 - 4 mice per group and represented as mean ± S . E . M

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(B) Flow cytometry plots of neutrophils and eosinophils in lungs at days 9 and 15 post sub-lethal influenza virus infection. Numbers in plots indicate frequency of gated immune cells in each throughput Data information: Data are representative of three independent experiments with 3-4 mice per group and represented as mean ± S.E.M


Mice were superinfected as described in ( A ) ( B ) Survival of mice following bacterial superinfection Data information : Data from ( B ) are combined from two independent experiments , n = 13 mice per experimental group All data are represented as mean ± S . E . M . Statistical significance was determined by logrank test in (

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Mice were superinfected as described in (A) (B) Survival of mice following bacterial superinfection Data information: Data from (B) are combined from two independent experiments, n=13 mice per experimental group All data are represented as mean ± S.E.M. Statistical significance was determined by logrank test in (B


Mice were superinfected as described in ( A ) ( C ) Pneumococcal cfu in BAL of mice Data information Dat are representative of at least two independent experiments with 3 - mice per experimental group All data are represented as mean ± S . E . M Statistical significance wa determined b Mann - Whitney U test in (

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Mice were superinfected as described in (A) (C) Pneumococcal cfu in BAL of mice Data information Dat are representative of at least two independent experiments with 3- mice per experimental group All data are represented as mean ± S.E.M Statistical significance wa determined b Mann-Whitney U test in (C


Mice were superinfected as described in ( A ) ( D ) Absolute immune cell counts in lungs , assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3 - mice per experimental group . All data are represented as mean ± S . E . M . Statistical significanc determined b unpaired student ' s T - test in (

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Mice were superinfected as described in (A) (D) Absolute immune cell counts in lungs, assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3- mice per experimental group. All data are represented as mean ± S.E.M. Statistical significanc determined b unpaired student's T-test in (D


Mice were superinfected as described in ( A ) ( E ) Pneumococcal cfu in BAL of mice 12 h after infection with 2x105 cfu of S . pneumoniae serotype 19F alone ( - Flu ) or following influenza virus infection ( + Flu ) Data information Dat are representative of at least two independent experiments wit 5 - mice per experimental group . All data are represented as mean ± S . E . M . Statistical significance was determined b Mann - Whitney U tes n . s . = not

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Mice were superinfected as described in (A) (E) Pneumococcal cfu in BAL of mice 12 h after infection with 2x105 cfu of S. pneumoniae serotype 19F alone (-Flu) or following influenza virus infection (+Flu) Data information Dat are representative of at least two independent experiments wit 5- mice per experimental group. All data are represented as mean ± S.E.M. Statistical significance was determined b Mann-Whitney U tes n.s. = not significant


Mice were superinfected as described in ( A ) ( F ) Pneumococcal cfu in BAL 36 h after infection of naïve mice with 2x105 cfu of S . pneumoniae serotype 19F alone Data information Dat are representative of at least two independent experiments wit 5 - mice per experimental group . All data are represented as mean ± S . E .

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Mice were superinfected as described in (A) (F) Pneumococcal cfu in BAL 36 h after infection of naïve mice with 2x105 cfu of S. pneumoniae serotype 19F alone Data information Dat are representative of at least two independent experiments wit 5- mice per experimental group. All data are represented as mean ± S.E.M


Mice were superinfected as described in ( A ) ( G ) Absolute neutrophil and eosinophil cell count in lungs infected as described in ( E ) , assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3 - mice per experimental group All data are represented as mean ± S . E . M . Statistical significance wa determined b ) , unpaired student ' s T - test i ( G ( E ) . n . s . = not

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Mice were superinfected as described in (A) (G) Absolute neutrophil and eosinophil cell count in lungs infected as described in (E), assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3- mice per experimental group All data are represented as mean ± S.E.M. Statistical significance wa determined b ), unpaired student's T-test i (G (E). n.s. = not significant


( A ) Concentrations of cytokines or chemokines in the BAL of days 5 and 9 post sub - lethal influenza virus infection as determined by protein bead array Data information : Data in ( A ) are obtained from one independent experiment with 4 mice per group , and results of selected analytes IFNγ , CXCL10 , IL - 6 , IL - 13 and IL - 17 are reconfirmed in a separate independent experiment by a customized protein bead array Significance i was determined by unpaired student ' s T - test . n . s . = not

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(A) Concentrations of cytokines or chemokines in the BAL of days 5 and 9 post sub-lethal influenza virus infection as determined by protein bead array Data information: Data in (A) are obtained from one independent experiment with 4 mice per group, and results of selected analytes IFNγ, CXCL10, IL-6, IL-13 and IL-17 are reconfirmed in a separate independent experiment by a customized protein bead array Significance i was determined by unpaired student's T-test. n.s. = not significant


Mice were superinfected as described in Fig 3A . ( B ) Concentrations of IL - 17 and IL - 22 in BAL of mice , as determined by ELISA Data informatio Data in ( B are representative of two independent experiments with 3 - 4 mice per group . All data are represented as mean ± S . E . M . Significance i was determined by unpaired student ' s T - test . n . s . = not

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Mice were superinfected as described in Fig 3A. (B) Concentrations of IL-17 and IL-22 in BAL of mice, as determined by ELISA Data informatio Data in (B are representative of two independent experiments with 3-4 mice per group. All data are represented as mean ± S.E.M. Significance i was determined by unpaired student's T-test. n.s. = not significant


Mice were superinfected as described in Fig 3A ( C ) Concentrations of IL - 17 and IL - 22 in BAL of mice 12 h after with 2x105 cfu of S . pneumoniae serotype 19F alone ( - Flu ) , or following superinfection ( + Flu ) , assessed by ELISA Data information Dat i ( C ) are representative of two independent experiments with 3 - 4 mice per group . All data are represented as mean ± S . E . M . Significanc was determined by unpaired student ' s T - test . n . s . = not

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Mice were superinfected as described in Fig 3A (C) Concentrations of IL-17 and IL-22 in BAL of mice 12 h after with 2x105 cfu of S. pneumoniae serotype 19F alone (-Flu), or following superinfection (+Flu), assessed by ELISA Data information Dat i (C) are representative of two independent experiments with 3-4 mice per group. All data are represented as mean ± S.E.M. Significanc was determined by unpaired student's T-test. n.s. = not significant


Mice were superinfected as described in Fig 3A . ( A ) Bacterial load as determined by enumeration of cfu in BAL following administration of αIL - 17 or αIL - 22 blocking antibody . Control wild - type and T - bet - / - groups were administered with IgG isotype control antibody Data information Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) All data are represented as mean ± S . E . M . Statistical significance was determined with Kruskal - Wallis test followed by Dunn " s multiple comparisons test n . s . = not

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Mice were superinfected as described in Fig 3A. (A) Bacterial load as determined by enumeration of cfu in BAL following administration of αIL-17 or αIL-22 blocking antibody. Control wild-type and T-bet-/- groups were administered with IgG isotype control antibody Data information Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined with Kruskal-Wallis test followed by Dunn"s multiple comparisons test n.s. = not significant


Mice were superinfected as described in Fig 3A ( B ) T - bet - / - mice were administered αIL - 17 or IgG isotype control antibody every other day from day - 2 post - secondary bacterial infection and monitored for survival Data information : Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) All data are represented as mean ± S . E . M . Statistical significance was determined wit logrank test in (

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Mice were superinfected as described in Fig 3A (B) T-bet-/- mice were administered αIL-17 or IgG isotype control antibody every other day from day -2 post-secondary bacterial infection and monitored for survival Data information: Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined wit logrank test in (B


Mice were superinfected as described in Fig 3A ( C ) Absolute neutrophil counts in the lungs following administration of αIL - 17 or IgG isotype control antibody , as determined by flow cytometry Data information Dat are representative of two independent experiments with 3 - 4 in ( C mice per group All data are represented as mean ± S . E . M . Statistical significance was determined wit unpaired - students t - test in (

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Mice were superinfected as described in Fig 3A (C) Absolute neutrophil counts in the lungs following administration of αIL-17 or IgG isotype control antibody, as determined by flow cytometry Data information Dat are representative of two independent experiments with 3-4 in (C mice per group All data are represented as mean ± S.E.M. Statistical significance was determined wit unpaired-students t-test in (C


Mice were superinfected as described in Fig 3A ( D ) Pneumococcal cfu in BAL following αLy6G antibody mediated neutrophil depletion . Control T - bet - / - mice were administered with IgG isotype control antibody Data information : Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) All data are represented as mean ± S . E . M . Statistical significance was determined wit Mann - Whitney U test in (

Mice were superinfected as described in Fig 3A (D) Pneumococcal cfu in BAL following αLy6G antibody mediated neutrophil depletion. Control T-bet-/- mice were administered with IgG isotype control antibody Data information: Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined wit Mann-Whitney U test in (D


Mice were superinfected as described in Fig 3A ( E ) mRNA expression of antimicrobial peptides in lungs following administration of αIL - 17 , IL - 22 or IgG control antibody , as determined by qPCR Data information Dat are representative of two independent experiments wit 4 - 6 in ( E ) mice per group . All data are represented as mean ± S . E . M . Statistical significance was determined wit one - way ANOVA followed by Tukey " s multiple comparisons test in ( E ) . n . s . = not

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Mice were superinfected as described in Fig 3A (E) mRNA expression of antimicrobial peptides in lungs following administration of αIL-17, IL-22 or IgG control antibody, as determined by qPCR Data information Dat are representative of two independent experiments wit 4-6 in (E) mice per group. All data are represented as mean ± S.E.M. Statistical significance was determined wit one-way ANOVA followed by Tukey"s multiple comparisons test in (E). n.s. = not signif


Mice were superinfected as described in Fig 3A ( A ) Frequency of IFNγ + and IL - 17 + immune cells following intracellular cytokine staining and flow cytometry Data information : Data are representative of at least two independent experiments with 3 mice per group . All data are represented as mean ± S . E . M . Statistical significanc was determined by unpaired student ' s T -

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Mice were superinfected as described in Fig 3A (A) Frequency of IFNγ+ and IL-17+ immune cells following intracellular cytokine staining and flow cytometry Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M. Statistical significanc was determined by unpaired student's T-test


Mice were superinfected as described in Fig 3A ( B ) Representative flow cytometry plots of total and IL - 17 + lymphocyte subsets on day 3 post - secondary bacterial infected lungs following intracellular cytokine staining . Numbers in plots indicate the frequency of each IL - 17 + cell subset over total numbers of respective cell - types Data information : Data are representative of at least two independent experiments with 3 mice per group . All data are represented as mean ± S . E . M

Mice were superinfected as described in Fig 3A (B) Representative flow cytometry plots of total and IL-17+ lymphocyte subsets on day 3 post-secondary bacterial infected lungs following intracellular cytokine staining. Numbers in plots indicate the frequency of each IL-17+ cell subset over total numbers of respective cell-types Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M


Mice were superinfected as described in Fig 3A ( C ) Frequency over total IL - 17 + cells and absolute numbers of each IL - 17 producing lymphocyte subset on days 0 , 1 and 3 post - secondary bacterial superinfected lungs as assessed by flow cytometry of intracellular cytokine stained cells Data information : Data are representative of at least two independent experiments with 3 mice per group . All data are represented as mean ± S . E . M . Statistical significanc i ( C ) was determined by unpaired student ' s T -

Mice were superinfected as described in Fig 3A (C) Frequency over total IL-17+ cells and absolute numbers of each IL-17 producing lymphocyte subset on days 0, 1 and 3 post-secondary bacterial superinfected lungs as assessed by flow cytometry of intracellular cytokine stained cells Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M. Statistical significanc i (C) was determined by unpaired student's T-test


B . Semi - quantitative PCR of miR - 132 and negative control miRNAs in the hippocampus of antagomiR - 132 - injected mice ( ant - 132 ) in comparison to control - injected animals ( aCSF and scramble ) at six months of age . Sample size , n = 9 per group .

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B. Semi-quantitative PCR of miR-132 and negative control miRNAs in the hippocampus of antagomiR-132-injected mice (ant-132) in comparison to control-injected animals (aCSF and scramble) at six months of age. Sample size, n=9 per group.


C . FISH of miR - 132 and negative control miR - 124 in the hippocampus of ant - 132 - and scramble - injected mice . Scramble probes were used as FISH negative controls . Scale bar , 500μm .

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C. FISH of miR-132 and negative control miR-124 in the hippocampus of ant-132- and scramble-injected mice. Scramble probes were used as FISH negative controls. Scale bar, 500μm.


D . Semi - quantitative PCR of miR - 132 and miR - 212 in the hippocampus of 3 - month old miR - 132 mimic - injected mice ( miR - 132 ) compared to animals injected with a negative control oligonucleotide ( Ctr ) . Sample size , n = 6 per group .

D. Semi-quantitative PCR of miR-132 and miR-212 in the hippocampus of 3-month old miR-132 mimic-injected mice (miR-132) compared to animals injected with a negative control oligonucleotide (Ctr). Sample size, n=6 per group.


A . Amyloid staining ( 6E10 ) combined with miR - 132FISH in ant - 132 - and scramble - injected mice at six months of age . Scale bar , 500 μm . B . Quantification of amyloid burden in hippocampus and cortex of ant - 132 - injected and control animals ( see Materials and Methods ) . Sample size , n = 4 per group .

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A. Amyloid staining (6E10) combined with miR-132FISH in ant-132- and scramble-injected mice at six months of age. Scale bar, 500 μm. B. Quantification of amyloid burden in hippocampus and cortex of ant-132-injected and control animals (see Materials and Methods). Sample size, n=4 per group.


C , D . ELISA of soluble ( TBS ) and insoluble ( Formic acid ) Aβ40 and Aβ42 levels in the hippocampus of ant - 132 - ( C ) and miR - 132 - injected ( D ) animals at six and three months of age , respectively . Sample size , n = 6 per group .

C, D. ELISA of soluble (TBS) and insoluble (Formic acid) Aβ40 and Aβ42 levels in the hippocampus of ant-132- (C) and miR-132-injected (D) animals at six and three months of age, respectively. Sample size, n=6 per group.


Western blot analysis of total TAU and pTAU ( AT8 , AT270 ) levels upon miR - 132 knockdown ( A ) or overexpression ( B ) at six and three months of age , respectively . Sample size , n = 9 per group ( A ) and n = 6 per group ( B ) . Values were normalized to the respective control groups and presented as mean ± SEM . Student ' s t - test was used .

Western blot analysis of total TAU and pTAU (AT8, AT270) levels upon miR-132 knockdown (A) or overexpression (B) at six and three months of age, respectively. Sample size, n=9 per group (A) and n=6 per group (B). Values were normalized to the respective control groups and presented as mean ± SEM. Student's t-test was used.


B . Positive hits were validated by semi - quantitative qPCR in ant - 132 - injected mousehippocampus . Black line indicates mean of scramble control set to 1 . Dashed line indicates chosen threshold of 1 . 3 - fold . Sample size , n = 9 per group . Values were normalized to scramble - injected group and presented as mean ± SEM .

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B. Positive hits were validated by semi-quantitative qPCR in ant-132-injected mousehippocampus. Black line indicates mean of scramble control set to 1. Dashed line indicates chosen threshold of 1.3-fold. Sample size, n=9 per group. Values were normalized to scramble-injected group and presented as mean ± SEM.


A . Luciferasereporter assay of wild - type ( wt ) and mutant ( mut ) ITPKB 3 ' UTR in HEK293 cells co - transfected with a synthetic miR - 132 ( miR - 132 ) or a negative control ( Neg Ctr ) oligonucleotide .

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A. Luciferasereporter assay of wild-type (wt) and mutant (mut) ITPKB 3'UTR in HEK293 cells co-transfected with a synthetic miR-132 (miR-132) or a negative control (Neg Ctr) oligonucleotide.


B . ELISA of Aβ40 and Aβ42 levels in HEK293 - APPswecells transfected with a miR - 132 antisense oligonucleotide ( miR - 132 inh ) , an siRNA against ITPKB ( ITPKB siRNA ) or both . The assays in A and B were performed in three independent experiments , each in triplicates .

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B. ELISA of Aβ40 and Aβ42 levels in HEK293-APPswecells transfected with a miR-132 antisense oligonucleotide (miR-132 inh), an siRNA against ITPKB (ITPKB siRNA) or both. The assays in A and B were performed in three independent experiments, each in triplicates.


C . Western blot analysis of ITPKB knock down in APPPS1hippocampus using an siRNA oligonucleotide against ITPKB . Sample size , n = 6 per group . Values were normalized to control - injected group ( Ctr ) and presented as mean ± SEM .

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C. Western blot analysis of ITPKB knock down in APPPS1hippocampus using an siRNA oligonucleotide against ITPKB. Sample size, n=6 per group. Values were normalized to control-injected group (Ctr) and presented as mean ± SEM.


D . ELISA of insoluble ( Formic acid - soluble ) Aβ40 and Aβ42 levels in the hippocampus of ITPKB siRNA - and control - injected animals at three months of age . Sample size , n = 6 per group . Values were normalized to control group and presented as mean ± SEM .

D. ELISA of insoluble (Formic acid-soluble) Aβ40 and Aβ42 levels in the hippocampus of ITPKB siRNA- and control-injected animals at three months of age. Sample size, n=6 per group. Values were normalized to control group and presented as mean ± SEM.


E , F . Western blot analysis of ITPKB levels upon miR - 132 down - ( E ) or up - regulation ( F ) at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to control groups and presented as mean ± SEM .

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E, F. Western blot analysis of ITPKB levels upon miR-132 down- (E) or up-regulation (F) at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to control groups and presented as mean ± SEM.


Western blot analysis of CTFβ ( A ) levels upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups and presented as mean ± SEM . The Student ' s t - test was used .

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Western blot analysis of CTFβ (A) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.


Western blot analysis of sAPPβ - swe ( B ) levels upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups and presented as mean ± SEM . The Student ' s t - test was used .

Western blot analysis of sAPPβ-swe (B) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.


Western blot analysis of phosphorylated ERK1 / 2 ( pERK1 / 2 ) ( C ) levels upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups and presented as mean ± SEM . The Student ' s t - test was used .

Western blot analysis of phosphorylated ERK1/2 (pERK1/2) (C) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.


A . Double immunofluorescence staining of amyloid plaques ( 6E10 ) and ITPKB in AD prefrontal cortex . Scale bar , 50 μm . Arrowheads indicate ITPKB immunopositivity .

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A. Double immunofluorescence staining of amyloid plaques (6E10) and ITPKB in AD prefrontal cortex. Scale bar, 50 μm. Arrowheads indicate ITPKB immunopositivity.


B . miR - 132FISH coupled with double immunofluorescence against hyperphosphorylated TAU ( AT8 ) - containing neurofibrillary tangles ( NFTs ) and ITPKB in AD prefrontal cortex . miR - 124 was used as a control . Scale bar , 50 μm .

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B. miR-132FISH coupled with double immunofluorescence against hyperphosphorylated TAU (AT8)-containing neurofibrillary tangles (NFTs) and ITPKB in AD prefrontal cortex. miR-124 was used as a control. Scale bar, 50 μm.


C . Quantification of miR - 132 , ITPKB and hyperphosphorylated TAU ( pTAU ) signal in single neurons . Integrated intensity values of each signal per cell were normalized to the mean integrated density of each signal across all the cells analyzed ( see Materials and Methods ) . Sample size ( AD patients ) , n = 3 . Values are presented as mean ± SEM . Two - way Anova was used . Quantifications are summarized in the table provided . Arrow directions refer to the comparison of each normalized signal to the same signal in the other group ( in the " low miR - 132 " group comparisons are made to the " high miR - 132 " group and vice versa ) .

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C. Quantification of miR-132, ITPKB and hyperphosphorylated TAU (pTAU) signal in single neurons. Integrated intensity values of each signal per cell were normalized to the mean integrated density of each signal across all the cells analyzed (see Materials and Methods). Sample size (AD patients), n=3. Values are presented as mean ± SEM. Two-way Anova was used. Quantifications are summarized in the table provided. Arrow directions refer to the comparison of each normalized signal to the same signal in the other group (in the "low miR-132" group comparisons are made to the "high miR-132" group and vice versa).


Semi - quantitative PCR of miR - 132 and western blot analysis of ITPKB , phosphorylated ( pERK1 / 2 ) and total ERK1 / 2 , phosphorylated ( AT8 , AT270 ) and total TAU , phosphorylated sphingosine kinase 1 ( pSphK1 ) , BACE1 , full length APP ( flAPP ) , APP CTFs and sAPPβ in human AD hippocampi ( AD ) compared to non demented control samples ( ND ) . Sample size , n = 39 for AD and n = 15 for ND . Values were normalized to ND and presented as mean ± SEM . The Student ' s t - test was used .

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Semi-quantitative PCR of miR-132 and western blot analysis of ITPKB, phosphorylated (pERK1/2) and total ERK1/2, phosphorylated (AT8 , AT270) and total TAU, phosphorylated sphingosine kinase 1 (pSphK1), BACE1, full length APP (flAPP), APP CTFs and sAPPβ in human AD hippocampi (AD) compared to non demented control samples (ND). Sample size, n=39 for AD and n=15 for ND. Values were normalized to ND and presented as mean ± SEM. The Student's t-test was used.


( B ) Phenotypes of affected individuals . Camptodactyly , absent flexion crease , and limited forearm supination were observed .

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(B) Phenotypes of affected individuals. Camptodactyly, absent flexion crease, and limited forearm supination were observed.


( C - E ) T1 weighted MRI scan on upper limbs of subject IV : 7 . ( C ) The pronator quadratus absence of affected side was indicated by a red arrow . ( D ) No difference was found in palmar muscles . ( E ) Loss of lumbrcalis and interosseus muscles of fifth finger of affected side was indicated by a red arrow . ( F - H ) T1 weighted MRI scan on upper limbs of subject IV : 8 . ( F ) Increased epimysial fat was indicated by red arrow . ( G ) Completely loss of thenar eminences of both hands were indicated by red arrows . ( H ) Loss of radial lumbrcalis and interosseus muscles of both hands were indicated by red arrows .

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(C-E) T1 weighted MRI scan on upper limbs of subject IV:7. (C) The pronator quadratus absence of affected side was indicated by a red arrow. (D) No difference was found in palmar muscles. (E) Loss of lumbrcalis and interosseus muscles of fifth finger of affected side was indicated by a red arrow. (F-H) T1 weighted MRI scan on upper limbs of subject IV:8. (F) Increased epimysial fat was indicated by red arrow. (G) Completely loss of thenar eminences of both hands were indicated by red arrows. (H) Loss of radial lumbrcalis and interosseus muscles of both hands were indicated by red arrows.


( I ) The MET variants by Sanger sequencing was indicated by red arrow .

(I) The MET variants by Sanger sequencing was indicated by red arrow.


( J ) 293T cells were transfected with FLAG - tagged MET / METMut / Vector plasmids , and 48 h post transfection , cells were treated with 10 ng / mL recombinant human HGF for 1 h . Then MET / METMut protein purification and tyrosine kinase assay were conducted . Western Blot pictures representative of n = 3 experiments . METMut means p . Y1234C mutant MET . EGFR means epidermal growth factor receptor and serves as a positive control . NC means negative control .

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(J) 293T cells were transfected with FLAG-tagged MET/METMut/Vector plasmids, and 48 h post transfection, cells were treated with 10 ng/mL recombinant human HGF for 1 h. Then MET/METMut protein purification and tyrosine kinase assay were conducted. Western Blot pictures representative of n = 3 experiments. METMut means p.Y1234C mutant MET. EGFR means epidermal growth factor receptor and serves as a positive control. NC means negative control.


( A ) Gross appearance of wild - type and heterozygous newborns . ( B ) Graph showing weight of P0 mice . Sample size : WT ( n = 4 ) and Hetero ( n = 5 ) . * P < 0 . 05 , by one - way ANOVA and follow by Dunnett ' s post hoc test .

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(A) Gross appearance of wild-type and heterozygous newborns. (B) Graph showing weight of P0 mice. Sample size: WT (n = 4) and Hetero (n = 5). *P < 0.05, by one-way ANOVA and follow by Dunnett's post hoc test.


Sections of spine from wild types and heterozygotes at P0 , were conducted with HE staining and immunofluorescence staining using anti - myosin heavy chain antibody . Scale bars , 100 μm .

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Sections of spine from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm.


Sections of forelimbs , hindlimbs , hand , and foot from wild types and heterozygotes at P0 , were conducted with HE staining and immunofluorescence staining using anti - myosin heavy chain antibody . Scale bars , 100 μm .

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Sections of forelimbs, hindlimbs, hand, and foot from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm.


( H ) The mean number of myofibers in the muscles of the spine , forelimb , hindlimb , hand and foot was qualified . NS means no statistic significance . * P < 0 . 05 , * * P < 0 . 01 , by two - tailed independent student ' s t test . In ( C - H ) , n = 3 .

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(H) The mean number of myofibers in the muscles of the spine, forelimb, hindlimb, hand and foot was qualified. NS means no statistic significance. *P < 0.05, **P < 0.01, by two-tailed independent student's t test. In (C-H), n = 3.


( I ) Transmission electron microscope analysis of gastrocnemius from wild - type and heterozygous newborns . Black arrows denote mitochodria . Scale bars , 2 μm . WT means wild types , Hetero means heterozygotes .

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(I) Transmission electron microscope analysis of gastrocnemius from wild-type and heterozygous newborns. Black arrows denote mitochodria. Scale bars, 2 μm. WT means wild types, Hetero means heterozygotes.


( A ) In situ hybridization of E10 . 5 embryos using Pax3 probe . Pax3 expression ( brown signal ) was observed in limb bud and demomyotome ( DM ) , respectively . Cells labeled with Pax3 were indicated by red arrow . Scale bars , 200 μm .

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(A) In situ hybridization of E10.5 embryos using Pax3 probe. Pax3 expression (brown signal) was observed in limb bud and demomyotome (DM), respectively. Cells labeled with Pax3 were indicated by red arrow. Scale bars, 200 μm.


( B ) Gross appearance of E14 . 5 embryos of indicated genotype . Scale bars , 2 . 5 mm .

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(B) Gross appearance of E14.5 embryos of indicated genotype. Scale bars, 2.5 mm.


( C ) HE staining of forelimb , hindlimb , and paraspinal muscle of E14 . 5 embryos . Scale bars , 100 μm .

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(C) HE staining of forelimb, hindlimb, and paraspinal muscle of E14.5 embryos. Scale bars, 100 μm.


( D ) Gross appearance of E16 . 5 embryos of indicated genotype . Scale bars , 2 . 5 mm .

(D) Gross appearance of E16.5 embryos of indicated genotype. Scale bars, 2.5 mm.


( E ) HE staining of forelimb , hindlimb , and paraspinal muscle of E16 . 5 embryos . n = 3 , scale bars , 200 μm .

(E) HE staining of forelimb, hindlimb, and paraspinal muscle of E16.5 embryos. n = 3, scale bars, 200 μm.


( F ) Anti - Ki67 antibody was used to label proliferative myoblasts ( red fluorescence ) with DAPI labeled nucleuses ( blue fluorescence ) in paraspinal muscle . Scale bar , 25 μm . ( G ) Bar graph showing statistical analysis of positive rate of Ki67 - labeled nuclei , n = 3 with more than 150 cells analyzed per n , * * P < 0 . 01 , by chi - square test ( χ2 test ) . WT means wild types , Hetero means heterozygotes , and Homo means homozygotes .

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(F) Anti-Ki67 antibody was used to label proliferative myoblasts (red fluorescence) with DAPI labeled nucleuses (blue fluorescence) in paraspinal muscle. Scale bar, 25 μm. (G) Bar graph showing statistical analysis of positive rate of Ki67-labeled nuclei, n = 3 with more than 150 cells analyzed per n, **P < 0.01, by chi-square test (χ2 test). WT means wild types, Hetero means heterozygotes, and Homo means homozygotes.


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 .

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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 .

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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 (

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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


LDH release was measured after cold stress for 24 h . FCCP ( 200 µM ) , antimycin A ( 50 µM ) , mitoQ and decylQ ( 500 nM ) were used as pretreatments for 30 min . Data are presented as mean ± SEM ; n = 4 ( G ) , biological replicates , * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , by one - way ANOVA followed by Dunnett ' s or Bonferroni ' s multiple comparison test . whose raw value were normalized by the C11 - BODIPY 581 / 591 without cold stress of non - treatment , are presented as mean ± SEM ; n = 5 , biological replicates . * * p < 0 . 01 , * p < 0 . 05 , by one - way ANOVA followed by Dunnett ' s multiple comparison test .

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LDH release was measured after cold stress for 24 h. FCCP (200 µM), antimycin A (50 µM), mitoQ and decylQ (500 nM) were used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 4 (G) , biological replicates, ****p < 0.0001, ***p < 0.001, **p < 0.01, by one-way ANOVA followed by Dunnett's or Bonferroni's multiple comparison test. whose raw value were normalized by the C11-BODIPY 581/591 without cold stress of non-treatment, are presented as mean ± SEM; n = 5, biological replicates. **p < 0.01, *p < 0.05, by one-way ANOVA followed by Dunnett's multiple comparison test.


LDH release was measured after cold stress for 24 h . BAPTA - AM ( 10 µM ) was used as pretreatments for 30 min . Data are presented as mean ± SEM ; n = 3 , biological replicates , * * * * p < 0 . 0001 , by unpaired T - test .

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LDH release was measured after cold stress for 24 h. BAPTA-AM (10 µM) was used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 3, biological replicates, ****p < 0.0001, by unpaired T-test.


LDH release was measured after cold stress for 16 h . Data are presented as mean ± SEM ; n = 5 , biological replicates , * p < 0 . 05 , by unpaired T - test .

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LDH release was measured after cold stress for 16 h. Data are presented as mean ± SEM; n = 5, biological replicates, *p < 0.05, by unpaired T-test.


A - B ) Western - blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated with DMSO or 1 µM rapamycin ( Rapa ) for the indicated time periods ( A ) ( n = 7 ) or for 5 h with the indicated concentrations ( B ) ( n = 6 ) . One hour before harvesting , 100 nM bafilomycin A1 was added . Upper panels : representative Western blots ; lower panels : quantification of the LC3 - II / GAPDH ratio .

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A-B) Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated with DMSO or 1 µM rapamycin (Rapa) for the indicated time periods (A) (n = 7) or for 5 h with the indicated concentrations (B) (n = 6). One hour before harvesting, 100 nM bafilomycin A1 was added. Upper panels: representative Western blots; lower panels: quantification of the LC3-II/GAPDH ratio.


C ) GFP - LC3 - punctae quantification in HeLa cells treated for 5 h with different concentrations of rapamycin . Left : representative pictures . The scale bar represents 10 µm . Concentrations are mentioned in the right lower corner . Right : Quantification of the number of punctae per cell ( n = 3 ) . * p < 0 . 05 , repeated measurements ANOVA .

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C) GFP-LC3-punctae quantification in HeLa cells treated for 5 h with different concentrations of rapamycin. Left: representative pictures. The scale bar represents 10 µm. Concentrations are mentioned in the right lower corner. Right: Quantification of the number of punctae per cell (n = 3). * p<0.05, repeated measurements ANOVA.


A ) Representative measurements ( n = 4 ) of cytosolic Ca2 + signals , displayed as Fura2 ratio ( F340 / F380 ) , showing the effect of 0 . 3 µM and 100 µM ATP , 1 µM thapsigargin ( Tg ) or 10 µM ionomycin ( Iono ) in intact HeLa cells treated with different concentrations of rapamycin ( Rapa ) for 5 h . 45 s prior to the addition of ATP , Tg or Iono , EGTA ( 3 mM ) was given to buffer extracellular Ca2 + as indicated .

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A) Representative measurements (n = 4) of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 0.3 µM and 100 µM ATP, 1 µM thapsigargin (Tg) or 10 µM ionomycin (Iono) in intact HeLa cells treated with different concentrations of rapamycin (Rapa) for 5 h. 45 s prior to the addition of ATP, Tg or Iono, EGTA (3 mM) was given to buffer extracellular Ca2+ as indicated.


B ) Quantification of the average amplitude of the response ( F − F0 ) ( n = 4 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , repeated measurements ANOVA .

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B) Quantification of the average amplitude of the response (F−F0) (n = 4). * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.


C ) Mean resting cytosolic [ Ca2 + ] , measured in Fura2 - loaded HeLa cells treated with the indicated concentrations of rapamycin for 5 h , as well as in the absence ( Ctrl ) or presence of DMSO ( n = 3 ) . * p < 0 . 05 , repeated measurements ANOVA .

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C) Mean resting cytosolic [Ca2+], measured in Fura2-loaded HeLa cells treated with the indicated concentrations of rapamycin for 5 h, as well as in the absence (Ctrl) or presence of DMSO (n = 3). * p<0.05, repeated measurements ANOVA.


D ) Unidirectional 45Ca2 + - flux experiments in permeabilized cells pretreated with 1 µM rapamycin for 5 h or with DMSO ( Ctrl ) . Mean fractional 45Ca2 + release ( % / 2 min ) is shown as a function of time with the effect of 0 . 7 µM IP3 ( circles ) or no addition ( squares ) . The horizontal bar indicates the presence of IP3 .

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D) Unidirectional 45Ca2+-flux experiments in permeabilized cells pretreated with 1 µM rapamycin for 5 h or with DMSO (Ctrl). Mean fractional 45Ca2+ release (%/2 min) is shown as a function of time with the effect of 0.7 µM IP3 (circles) or no addition (squares). The horizontal bar indicates the presence of IP3.


E ) Quantitative analysis of the IP3 - induced 45Ca2 + release in cells pretreated for 5 h with 1 µM rapamycin or DMSO ( Ctrl ) ( n = 8 ) . * * * p < 0 . 001 , paired Student ' s t - test .

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E) Quantitative analysis of the IP3-induced 45Ca2+ release in cells pretreated for 5 h with 1 µM rapamycin or DMSO (Ctrl) (n = 8). *** p<0.001, paired Student's t-test.


A - B ) Western - blot analysis for luminal Ca2 + - binding proteins in HeLa cells treated with the indicated concentrations of rapamycin ( Rapa ) for 5 h : calreticulin ( CRT ) ( A ) and BiP / Grp78 ( BiP ) ( B ) . Upper panels : representative Western blots ; lower panels : quantification of the protein / GAPDH ratio ( n = 4 ) .

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A-B) Western-blot analysis for luminal Ca2+-binding proteins in HeLa cells treated with the indicated concentrations of rapamycin (Rapa) for 5 h: calreticulin (CRT) (A) and BiP/Grp78 (BiP) (B). Upper panels: representative Western blots; lower panels: quantification of the protein/GAPDH ratio (n = 4).


C ) Western - blot analysis for SERCA2 in HeLa cells treated with the indicated concentrations of rapamycin for 5 h . Upper panels : representative Western blots ; lower panel : quantification of the SERCA2 / GAPDH ratio ( n = 4 ) .

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C) Western-blot analysis for SERCA2 in HeLa cells treated with the indicated concentrations of rapamycin for 5 h. Upper panels: representative Western blots; lower panel: quantification of the SERCA2/GAPDH ratio (n = 4).


D ) Representative plot showing the decrease in ER 45Ca2 + content ( logarithmic scale ) in a Ca2 + - free efflux medium without ATP as a function of time in permeabilized HeLa cells pretreated for 5 h with 1 µM rapamycin or with DMSO . The passively bound Ca2 + was determined by loading the cells with 45Ca2 + in the presence of 10 µM of the Ca2 + ionophore A23187 and then subtracted from the stored 45Ca2 + . The ER Ca2 + - leak rate can be estimated as the rate of decline of the ER 45Ca2 + - store content as a function of time .

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D) Representative plot showing the decrease in ER 45Ca2+ content (logarithmic scale) in a Ca2+-free efflux medium without ATP as a function of time in permeabilized HeLa cells pretreated for 5 h with 1 µM rapamycin or with DMSO. The passively bound Ca2+ was determined by loading the cells with 45Ca2+ in the presence of 10 µM of the Ca2+ ionophore A23187 and then subtracted from the stored 45Ca2+. The ER Ca2+-leak rate can be estimated as the rate of decline of the ER 45Ca2+-store content as a function of time.


E ) Quantification of the mean 45Ca2 + - store content at the beginning of the measurement ( t0 ) ( n = 5 ) .

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E) Quantification of the mean 45Ca2+-store content at the beginning of the measurement (t0) (n = 5).


F ) Quantification of the mean slope of the curve in D after transformation to a linear scale , which is a measure of the 45Ca2 + - leak rate ( n = 5 ) . * p < 0 . 05 ; * * p < 0 . 01 , paired Student ' s t - test .

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F) Quantification of the mean slope of the curve in D after transformation to a linear scale, which is a measure of the 45Ca2+-leak rate (n = 5). * p<0.05; ** p<0.01, paired Student's t-test.


A ) Representative Western - blot analysis for Atg12 ( showing the autophagic Atg12 - Atg5 complex ) , GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with ( + Dox ) or without ( - Dox ) doxycycline and treated with DMSO or 0 . 1 , 1 or 5 µM rapamycin ( Rapa ) for 5 h ( n = 3 ) .

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A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h (n = 3).


B - C ) Representative measurements of cytosolic Ca2 + signals , displayed as Fura2 ratio ( F340 / F380 ) , showing the effect of 1 mM ATP ( B ) or 10 µM ionomycin ( Iono ) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h . Prior to the addition of ATP or Iono , EGTA ( 3 mM ) was added to chelate the extracellular Ca2 + as indicated .

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B-C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated.


D ) Quantification of the average amplitude of the response ( F − F0 ) ( n = 3 , 4 , 5 and 6 for ATP - Dox , ATP + Dox , Iono - Dox and Iono + Dox , resp . ) * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , repeated measurements ANOVA .

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D) Quantification of the average amplitude of the response (F−F0) (n = 3, 4, 5 and 6 for ATP-Dox, ATP+Dox, Iono-Dox and Iono+Dox, resp.) * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.


2 + - dependent . Western - blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated for 5 h with DMSO , 1 µM rapamycin ( Rapa ) , 10 µM BAPTA - AM or both . One hour before harvesting , 100 nM bafilomycin A1 was added . Left : representative Western blots ; right : quantification of the LC3 - II / GAPDH ratio ( n = 6 ) . * p < 0 . 05 , repeated measurements ANOVA .

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2+-dependent. Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated for 5 h with DMSO, 1 µM rapamycin (Rapa), 10 µM BAPTA-AM or both. One hour before harvesting, 100 nM bafilomycin A1 was added. Left: representative Western blots; right: quantification of the LC3-II/GAPDH ratio (n = 6). * p<0.05, repeated measurements ANOVA.


Average piRNA precursor abundance relative to EV in EV and ints - 11 RNAi samples , normalized to the total number of short capped RNA reads mapping to annotated WormBase TSSs ( Chen et al . , 2013 ) . Error bars represent the standard error of the mean ( 5 replicates ) .

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Average piRNA precursor abundance relative to EV in EV and ints-11 RNAi samples, normalized to the total number of short capped RNA reads mapping to annotated WormBase TSSs (Chen et al., 2013). Error bars represent the standard error of the mean (5 replicates).


Distributions of piRNA precursor length in ints - 11 RNAi - treated nematodes compared to empty vector - treated nematodes , obtained from deeply sequencing one pair of libraries ( inserts sequenced up to 75nt ) . Distributions of total sequences ( E ) and total read counts ( F ) are shown .

Distributions of piRNA precursor length in ints-11 RNAi-treated nematodes compared to empty vector-treated nematodes, obtained from deeply sequencing one pair of libraries (inserts sequenced up to 75nt). Distributions of total sequences (E) and total read counts (F) are shown.


Co - localization between INTS - 6 : : eGFP and mCherry : : PRDE - 1 in germ cells . PRDE - 1 and INTS - 6 co - localize in early stages of meiosis ( 1 , 2 ) and up to mid - pachytene ( 3 ) , where an additional INTS - 6 focus appears ( 3 ) . When PRDE - 1 foci are about to disappear , co - localization is lost ( 4 ) , and the additional INTS - 6 focus remains present well after the loss of PRDE - 1 foci ( 5 ) .

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Co-localization between INTS-6::eGFP and mCherry::PRDE-1 in germ cells. PRDE-1 and INTS-6 co-localize in early stages of meiosis (1,2) and up to mid-pachytene (3), where an additional INTS-6 focus appears (3). When PRDE-1 foci are about to disappear, co-localization is lost (4), and the additional INTS-6 focus remains present well after the loss of PRDE-1 foci (5).


Length distributions of nucleoplasmic and chromatin - bound piRNA precursors in N2 empty vector , N2 ints - 11 RNAi , tfiis empty vector , and tfiis ints - 11 RNAi nematodes . Length shifts in nascent RNA in tfiis mutants are indicated with dashed vertical lines and arrows . The length distributions were calculated from two merged replicates ( see Appendix Figure S2 for the length distributions in each replicate ) .

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Length distributions of nucleoplasmic and chromatin-bound piRNA precursors in N2 empty vector, N2 ints-11 RNAi, tfiis empty vector, and tfiis ints-11 RNAi nematodes. Length shifts in nascent RNA in tfiis mutants are indicated with dashed vertical lines and arrows. The length distributions were calculated from two merged replicates (see Appendix Figure S2 for the length distributions in each replicate).


Signal of 5 ′ P small RNA 5 ′ ends mapping to piRNA loci as a function of the distance to piRNA TSSs , after removal of reads corresponding to mature 21U - RNAs ( see Methods ) . The signal is normalized to counts per million of non - structural mapped reads . A peak of 5 ′ P ends centered at + 38 from piRNA TSSs is observed .

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Signal of 5′ P small RNA 5′ ends mapping to piRNA loci as a function of the distance to piRNA TSSs, after removal of reads corresponding to mature 21U-RNAs (see Methods). The signal is normalized to counts per million of non-structural mapped reads. A peak of 5′ P ends centered at +38 from piRNA TSSs is observed.


Number of loci with detected cleavage fragments in N2 empty vector nematodes across percentile bins of increasing termination signal strength ( top panel ) . Distributions of fragment read counts per locus for detected loci across percentile bins of increasing termination signal strength ( bottom panel ) . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile range . The data shown here corresponds to one of two N2 EV replicates , see Appendix Figure S6 for this analysis across replicates and genotypes .

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Number of loci with detected cleavage fragments in N2 empty vector nematodes across percentile bins of increasing termination signal strength (top panel). Distributions of fragment read counts per locus for detected loci across percentile bins of increasing termination signal strength (bottom panel). Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range. The data shown here corresponds to one of two N2 EV replicates, see Appendix Figure S6 for this analysis across replicates and genotypes.


Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif - dependent piRNAs upon ints - 11 RNAi knockdown in an N2 and a tfiis mutant background calculated from two merged replicates . N2 empty vector was used as a baseline for fold change calculation . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile range .

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Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif-dependent piRNAs upon ints-11 RNAi knockdown in an N2 and a tfiis mutant background calculated from two merged replicates. N2 empty vector was used as a baseline for fold change calculation. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range.


Average normalized CAGE signal ( transcripts per million ) 2 nt upstream of motif - dependent and motif - independent piRNAs ( excluding undetected loci ) . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile

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Average normalized CAGE signal (transcripts per million) 2 nt upstream of motif-dependent and motif-independent piRNAs (excluding undetected loci). Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range


Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif - independent piRNAs upon ints - 11 RNAi knockdown in an N2 and a tfiis mutant background . N2 empty vector was used as a baseline for fold change calculation . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile

Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif-independent piRNAs upon ints-11 RNAi knockdown in an N2 and a tfiis mutant background. N2 empty vector was used as a baseline for fold change calculation. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range


B . Deconvolved fluorescence micrographs of Chm7 - GFP and truncations with Nup170 - mCherry NE marker and merge of green and red channels . Scale bar is 5 μm .

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B. Deconvolved fluorescence micrographs of Chm7-GFP and truncations with Nup170-mCherry NE marker and merge of green and red channels. Scale bar is 5 μm.


C . Scatter plot of the mean fluorescence intensity with SD of individual NE foci ( horizontal line is mean ) . > 50 foci were measured for each GFP fusion ; p values from un - paired student ' s T - test . * * * is p ≤ 0 . 001 ; * * * * is p ≤ 0 . 0001 .

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C. Scatter plot of the mean fluorescence intensity with SD of individual NE foci (horizontal line is mean). >50 foci were measured for each GFP fusion; p values from un-paired student's T-test. *** is p ≤ 0.001; **** is p ≤ 0.0001.


D . Deconvolved fluorescence micrographs of Chm7 - GFP and Mps3 - mCherry with merge of green and red images ( right ) . Scale bar is 5 μm .

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D. Deconvolved fluorescence micrographs of Chm7-GFP and Mps3-mCherry with merge of green and red images (right). Scale bar is 5 μm.


E . 10 - fold serial dilutions of chm7Δapq12Δ strains grown at the indicated temperatures with either an empty pRS416 plasmid ( - ) or those expressing the indicated Chm7 constructs ( pRS416 - HA - CHM7 , pRS416 - HA - chm7 - NTD , pRS416 - HA - chm7 - CTD ) . See Appendix Figure S2A for protein levels .

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E. 10-fold serial dilutions of chm7Δapq12Δ strains grown at the indicated temperatures with either an empty pRS416 plasmid (-) or those expressing the indicated Chm7 constructs (pRS416-HA-CHM7, pRS416-HA-chm7-NTD, pRS416-HA-chm7-CTD). See Appendix Figure S2A for protein levels.


A . Deconvolved inverted fluorescence micrographs Chm7 - GFP and truncations in the indicated null strains . Scale bar is 5 μm . B . Plot of the percentage of cells with SD of Chm7 - GFP ( and truncations ) NE foci from A . Data are from 3 independent replicates where > 200 cells were counted for each strain . p values from un - paired student ' s T - test . * * is a p value ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 .

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A. Deconvolved inverted fluorescence micrographs Chm7-GFP and truncations in the indicated null strains. Scale bar is 5 μm.B. Plot of the percentage of cells with SD of Chm7-GFP (and truncations) NE foci from A. Data are from 3 independent replicates where >200 cells were counted for each strain. p values from un-paired student's T-test. ** is a p value ≤ 0.01; ****, p ≤ 0.0001.


C . Deconvolved inverted fluorescence micrographs of Chm7 - GFP in a heh1Δ strain containing either an empty pRS426 plasmid ( - ) or those expressing HEH1 ( pRS426 - HEH1 ) or HEH2 ( pRS426 - HEH2 ) . D . Plots of the percentage of cells from C with Chm7 - GFP NE - foci . Data are from 3 independent replicates where > 200 cells were counted for each strain . Error bars are SD . p values from T - test . * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 .

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C. Deconvolved inverted fluorescence micrographs of Chm7-GFP in a heh1Δ strain containing either an empty pRS426 plasmid (-) or those expressing HEH1 (pRS426-HEH1) or HEH2 (pRS426-HEH2).D. Plots of the percentage of cells from C with Chm7-GFP NE-foci. Data are from 3 independent replicates where >200 cells were counted for each strain. Error bars are SD. p values from T-test. **, p ≤ 0.01; ****, p ≤ 0.0001.


C . GST , GST - Snf7 and GST - snf7OPEN were immobilized on GT - resin and incubated with buffer ( - ) or recombinant heh2 ( 1 - 308 ) - His6 . Bound proteins separated by SDS - PAGE were visualized by coomassie stain and by Western blot with anti - His6 antibody and ECL detection ( WB ; top panel ) . Middle panel shows indicated cropped region of gel where contrast has been increased . Numbers on side of gel show position of molecular weight ( MW ) markers .

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C. GST, GST-Snf7 and GST-snf7OPEN were immobilized on GT-resin and incubated with buffer(-) or recombinant heh2(1-308)-His6. Bound proteins separated by SDS-PAGE were visualized by coomassie stain and by Western blot with anti-His6 antibody and ECL detection (WB; top panel). Middle panel shows indicated cropped region of gel where contrast has been increased. Numbers on side of gel show position of molecular weight (MW) markers.


D . In vitro transcription translation ( IVT ) reactions generating radiolabeled ( 35S ) truncations of Heh2 ( inputs ) were incubated with bead - bound GST , GST - snf7OPEN or GST - Snf7 before washing , elution and detection of bound proteins by autoradiography ( top panel ) or coomassie staining ( bottom ) .

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D. In vitro transcription translation (IVT) reactions generating radiolabeled (35S) truncations of Heh2 (inputs) were incubated with bead-bound GST, GST-snf7OPEN or GST-Snf7 before washing, elution and detection of bound proteins by autoradiography (top panel) or coomassie staining (bottom).


E . As in C except with GST - Chm7 constructs . Upper panel shows western blot ( WB ) with anti - His6 antibody . * chm7 - NTD is also expressed with a His6 tag . Bottom panel is coomassie stained .

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E. As in C except with GST-Chm7 constructs. Upper panel shows western blot (WB) with anti-His6 antibody. *chm7-NTD is also expressed with a His6 tag. Bottom panel is coomassie stained.


A . Deconvolved inverted fluorescent micrographs of BiFC signal of Chm7 - VC with either Heh1 - VN or Heh2 - VN . Cell borders are outlined . Scale bar is 5 μm .

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A. Deconvolved inverted fluorescent micrographs of BiFC signal of Chm7-VC with either Heh1-VN or Heh2-VN. Cell borders are outlined. Scale bar is 5 μm.


B . Deconvolved inverted fluorescent micrographs of yeast strains expressing Heh1 - VN and Snf7 - VC with either Chm7 - mCherry or Mps3 - mCherry and merge of both channels . Scale bar is 5 μm .

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B. Deconvolved inverted fluorescent micrographs of yeast strains expressing Heh1-VN and Snf7-VC with either Chm7-mCherry or Mps3-mCherry and merge of both channels. Scale bar is 5 μm.


C . Deconvolved inverted fluorescent micrographs of BiFC signal of the indicated VN and VC fusions in the indicated strains . Cell borders outlined . Scale bar is 5 μm .

C. Deconvolved inverted fluorescent micrographs of BiFC signal of the indicated VN and VC fusions in the indicated strains. Cell borders outlined. Scale bar is 5 μm.


D , E . Plots of the percentage of cells with BiFC signal of the indicated VN VC pairs in the indicated null backgrounds . Data are from three independent replicates where > 225 cells per strain per replicate were quantified . Error bars are SD from the mean of each replicate . p values from un - paired student ' s T - test where ns is p > 0 . 05 . * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 ; * * * * , p ≤ 0 . 0001 .

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D,E. Plots of the percentage of cells with BiFC signal of the indicated VN VC pairs in the indicated null backgrounds. Data are from three independent replicates where > 225 cells per strain per replicate were quantified. Error bars are SD from the mean of each replicate. p values from un-paired student's T-test where ns is p > 0.05. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.