Colitis was induced by oral administration of 3 % DSS . Mice were treated daily ( s . c . ) with LU - 001i ( 15 mg / kg ) , PRN1126 ( 40 mg / kg ) , PRN1126 + LU - 001i ( 40 mg / kg + 15 mg / kg ) , or vehicle starting from the begin of the experiment . Data points represent means ± SEM of 15 mice pooled from three independent experiments . On day 9 after initiation of DSS treatment , colon lengths were measured ( n = 15 ) . Naïve mice ( n = 5 ) were used as healthy controls . All data were statistically compared to the vehicle treated group .

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 B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Colitis was induced by oral administration of 3% DSS. Mice were treated daily (s.c.) with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the begin of the experiment. Data points represent means ± SEM of 15 mice pooled from three independent experiments. On day 9 after initiation of DSS treatment, colon lengths were measured (n=15). Naïve mice (n=5) were used as healthy controls. All data were statistically compared to the vehicle treated group.


Mice were immunized with MOG35 - 55 peptide and were monitored daily for clinical symptoms of EAE . Mice were treated intermittently with three times a week ( s . c . ) schedule with LU - 001i ( 15 mg / kg ) , PRN1126 ( 40 mg / kg ) , PRN1126 + LU - 001i ( 40 mg / kg + 15 mg / kg ) , or vehicle starting from the start of the experiment . Data points represent the means of the clinical scores ± SEM of 12 mice pooled from two independent experiments . All data were statistically compared to the vehicle treated group .

O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O

Mice were immunized with MOG35-55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated intermittently with three times a week (s.c.) schedule with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the start of the experiment. Data points represent the means of the clinical scores ± SEM of 12 mice pooled from two independent experiments. All data were statistically compared to the vehicle treated group.


C57BL / 6 , SHP - KO , and FGF15 - KO or littermate mice were fasted overnight and then re - fed for 6 ( A - C ) Levels of the indicated proteins detected by IB in ( A ) liver and intestinal extracts from C57BL / 6 mice and in intestinal extracts from ( B ) C57BL / 6 or SHP - KO mice or ( C ) FGF15 - KO mice or littermates . Band quantitation was done using Image J , and the LC3 II / I ratio and p62 / actin and lamp1 / actin ratios are shown at the right . ( A - C ) The mean and standard deviation ( SD ) are plotted . Statistical significance was determined by ( A ) two - tailed Student ' s t - tests or ( B , C ) two - way ANOVA with the Tukey post - test . P - values are indicated ( n = 3 mice ) .

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

C57BL/6, SHP-KO, and FGF15-KO or littermate mice were fasted overnight and then re-fed for 6 (A-C) Levels of the indicated proteins detected by IB in (A) liver and intestinal extracts from C57BL/6 mice and in intestinal extracts from (B) C57BL/6 or SHP-KO mice or (C) FGF15-KO mice or littermates. Band quantitation was done using Image J, and the LC3 II/I ratio and p62/actin and lamp1/actin ratios are shown at the right. (A-C) The mean and standard deviation (SD) are plotted. Statistical significance was determined by (A) two-tailed Student's t-tests or (B, C) two-way ANOVA with the Tukey post-test. P-values are indicated (n=3 mice).


C57BL / 6 , SHP - KO , and FGF15 - KO or littermate mice as indicated were fasted for 12 h and then , re - fed for 6 h . levels of the indicated proteins in intestinal extracts from ( B ) C57BL / 6 and SHP - KO mice or ( D ) FGF15 - KO mice and littermates with quantitation of the bands at right . , ( B , D ) n = 3 mice . The mean and SD are plotted . Statistical significance was determined by two - way ANOVA with the Tukey post - test P - values are

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

C57BL/6, SHP-KO, and FGF15-KO or littermate mice as indicated were fasted for 12 h and then, re-fed for 6 h. levels of the indicated proteins in intestinal extracts from (B) C57BL/6 and SHP-KO mice or (D) FGF15-KO mice and littermates with quantitation of the bands at right. , (B, D) n=3 mice. The mean and SD are plotted. Statistical significance was determined by two-way ANOVA with the Tukey post-test P-values are indicated


( G ) HT29 cells were transfected with GFP - LC3 and with SHP siRNA or SHP expression plasmids and incubated in serum - free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 6 h The control group ( Veh ) was treated with vehicle for FGF19 , control plasmid DNA , and scrambled siRNA . The FGF19 groups were treated with control DNA or SHP expression plasmid or SHP siRNA or control scrambled siRNA as indicated . Cells were imaged by confocal microscopy ( Zeiss , LSM700 ) . GFP - LC3 puncta were counted and the number of puncta / cell is shown at right ( n = 20 ) . Scale bar , 10 μm . G , The mean and SD are plotted . Statistical significance was determined by ( G ) one - way ANOVA with the Tukey post - test . P - values are

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were transfected with GFP-LC3 and with SHP siRNA or SHP expression plasmids and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h The control group (Veh) was treated with vehicle for FGF19, control plasmid DNA, and scrambled siRNA. The FGF19 groups were treated with control DNA or SHP expression plasmid or SHP siRNA or control scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20). Scale bar, 10 μm. G, The mean and SD are plotted. Statistical significance was determined by (G) one-way ANOVA with the Tukey post-test. P-values are indicated


( H ) HT29 cells were treated with 50 ng / ml FGF19 with or without 100 nM bafilomycin A1 for 12 h . LC3 and actin levels in cell extracts were determined by IB . Quantification of bands is shown at the right ( n = 3 culture plates ) . H ) The mean and SD are plotted . Statistical significance was determined by two - way ANOVA with the Tukey post - test P - values are

O O O O O O O O O O O O O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O

(H) HT29 cells were treated with 50 ng/ml FGF19 with or without 100 nM bafilomycin A1 for 12 h. LC3 and actin levels in cell extracts were determined by IB. Quantification of bands is shown at the right (n=3 culture plates). H) The mean and SD are plotted. Statistical significance was determined by two-way ANOVA with the Tukey post-test P-values are indicated


( I ) HT29 cells were transfected with Lamp1 siRNA or scrambled siRNA and 48 h later were treated with 50 ng / ml FGF19 for the indicated times . ApoB48 and actin levels were determined by IB ( n = 2 culture plates ) .

O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were transfected with Lamp1 siRNA or scrambled siRNA and 48 h later were treated with 50 ng/ml FGF19 for the indicated times. ApoB48 and actin levels were determined by IB (n=2 culture plates).


( A ) HT29 cells were transfected with GFP - LC3 and with Atg7 siRNA or control scrambled RNA ( siCtl ) and incubated in serum - free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 6 h The control group ( Veh ) was treated with vehicle for FGF19 and control siRNA . The FGF19 groups were treated with Atg7 siRNA or scrambled siRNA as indicated . Cells were imaged by confocal microscopy ( Zeiss , LSM700 ) . GFP - LC3 puncta were counted and the number of puncta / cell is shown at right ( n = 20 ) . Scale bar , 20 μm . The mean and SD are plotted . Statistical significance was determined by one - way ANOVA with the Tukey post - test . P - values are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were transfected with GFP-LC3 and with Atg7 siRNA or control scrambled RNA (siCtl) and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h The control group (Veh) was treated with vehicle for FGF19 and control siRNA. The FGF19 groups were treated with Atg7 siRNA or scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20). Scale bar, 20 μm. The mean and SD are plotted. Statistical significance was determined by one-way ANOVA with the Tukey post-test. P-values are indicated.


( B ) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA and 48 h later were treated with 50 ng / ml FGF19 for 12 h . LC3 and actin levels in cell extracts determined by IB ( n = 3 culture plates ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA and 48 h later were treated with 50 ng/ml FGF19 for 12 h. LC3 and actin levels in cell extracts determined by IB (n=3 culture plates).


D ) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA ( siCtl ) for 48 h and then treated with oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 12 h . ( D ) ApoB48 and actin levels in cell extracts determined by IB . Bands were quantified and the LC3 - II / LC3 - I ratio is shown at the right ( n = 3 culture plates ) . The mean and SD are plotted . Statistical significance was determined by one - way ANOVA with the Tukey post - test . P - values are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA (siCtl) for 48 h and then treated with oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 12 h. (D) ApoB48 and actin levels in cell extracts determined by IB. Bands were quantified and the LC3-II/LC3-I ratio is shown at the right (n=3 culture plates). The mean and SD are plotted. Statistical significance was determined by one-way ANOVA with the Tukey post-test. P-values are indicated.


Mice were re - fed for 2 h ( B ) after fasting overnight . ( B ) CoIP of TFEB with SHP . Upper panel : Levels of TFEB detected by IB in SHP and IgG immunoprecipitated samples . Lower panel : levels of TFEB and SHP in input samples detected by IB . Similar results were obtained in a second experiment .

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

Mice were re-fed for 2 h (B) after fasting overnight. (B) CoIP of TFEB with SHP. Upper panel: Levels of TFEB detected by IB in SHP and IgG immunoprecipitated samples. Lower panel: levels of TFEB and SHP in input samples detected by IB. Similar results were obtained in a second experiment.


( F , G ) Protein levels detected by IB of ULK1 and ATGL in intestinal extracts from ( F ) C57BL / 6 and SHP - KO mice or ( G ) FGF15 - KO mice or littermates re - fed for 6 h after fasting overnight ( n = 3 mice ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G) Protein levels detected by IB of ULK1 and ATGL in intestinal extracts from (F) C57BL/6 and SHP-KO mice or (G) FGF15-KO mice or littermates re-fed for 6 h after fasting overnight (n=3 mice).


( A ) HT29 cells were transfected with GFP - LC3 and with Tfeb siRNA or Tfeb expression plasmids and incubated in serum - free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 6 h The control group ( Veh ) was treated with vehicle for FGF19 and transfected with control plasmid DNA and scrambled siRNA . The FGF19 groups were treated with control DNA or TFEB expression plasmid or with Tfeb siRNA or scrambled siRNA as indicated . Cells were imaged by confocal microscopy ( Zeiss , LSM700 ) . GFP - LC3 puncta were counted and the number of puncta / cell is shown at right ( n = 20 cells ) . Scale bar , 10 μm . ( A , The mean and SD are plotted . Statistical significance was determined by ( A ) one - way ANOVA with the Tukey post - test , P - values are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were transfected with GFP-LC3 and with Tfeb siRNA or Tfeb expression plasmids and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h The control group (Veh) was treated with vehicle for FGF19 and transfected with control plasmid DNA and scrambled siRNA. The FGF19 groups were treated with control DNA or TFEB expression plasmid or with Tfeb siRNA or scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20 cells). Scale bar, 10 μm. (A, The mean and SD are plotted. Statistical significance was determined by (A) one-way ANOVA with the Tukey post-test, P-values are indicated.


( B ) HT29 cells were transfected with siRNA for Tfeb ( siTFEB ) or control scrambled siRNA ( siCtl ) , followed 48 h later by treatment with FGF19 or vehicle with or without 100 nM bafilomycin A1 ( BAF ) for 12 h . Levels of the indicated proteins determined by IB . LC3 II / I and p62 / actin ratios are shown at the right ( n = 3 - 4 culture plates ) . B , The mean and SD are plotted . Statistical significance was determined by ( B ) two - way ANOVA with the Tukey post - test , P - values are indicated .

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

(B) HT29 cells were transfected with siRNA for Tfeb (siTFEB) or control scrambled siRNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle with or without 100 nM bafilomycin A1 (BAF) for 12 h. Levels of the indicated proteins determined by IB. LC3 II/I and p62/actin ratios are shown at the right (n=3-4 culture plates). B, The mean and SD are plotted. Statistical significance was determined by (B) two-way ANOVA with the Tukey post-test, P-values are indicated.


( C ) HT29 cells were transfected with siRNA for TFEB ( siTFEB ) or control RNA ( siCtl ) , followed 48 h later by treatment with FGF19 or vehicle for 12 h . Protein levels detected by IB of ULK1 and ATGL ( n = 3 culture plates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were transfected with siRNA for TFEB (siTFEB) or control RNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle for 12 h. Protein levels detected by IB of ULK1 and ATGL (n=3 culture plates).


( E ) HT29 cells were transfected with siRNA for Tfeb ( siTFEB ) or scrambled siRNA ( siCtl ) , followed 48 h later by treatment with FGF19 or vehicle for 12 h . ApoB48 and actin levels in cell extracts determined by IB ( n = 3 culture plates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were transfected with siRNA for Tfeb (siTFEB) or scrambled siRNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle for 12 h. ApoB48 and actin levels in cell extracts determined by IB (n=3 culture plates).


( A , C57BL / 6 ( A ) were re - fed for 6 h after fasting overnight . The cytosolic and nuclear fractions were isolated from intestine or liver , and the indicated proteins were detected by IB ( n = 2 mice ) . TBLN indicates tubulin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C57BL/6 (A) were re-fed for 6 h after fasting overnight. The cytosolic and nuclear fractions were isolated from intestine or liver, and the indicated proteins were detected by IB (n=2 mice). TBLN indicates tubulin.


B ) FGF15 - KO mice or littermates ( B ) were re - fed for 6 h after fasting overnight . the indicated proteins were detected by IB ( n = 2 mice ) . TBLN indicates tubulin .

O O O O O O O O O O O O O O O O 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) FGF15-KO mice or littermates (B) were re-fed for 6 h after fasting overnight. the indicated proteins were detected by IB (n=2 mice). TBLN indicates tubulin.


FGF15 - KO mice or littermates were fasted overnight and re - fed for the indicated times . ( C ) The nuclear fraction was isolated and the nuclear levels of TFEB , SHP , and LAMIN were determined by IB .

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

FGF15-KO mice or littermates were fasted overnight and re-fed for the indicated times. (C) The nuclear fraction was isolated and the nuclear levels of TFEB, SHP, and LAMIN were determined by IB.


FGF15 - KO mice or littermates were fasted overnight and re - fed for the indicated times . ( D ) Levels of LC3 , p62 , and actin in cell extracts were determined by IB .

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

FGF15-KO mice or littermates were fasted overnight and re-fed for the indicated times. (D) Levels of LC3, p62, and actin in cell extracts were determined by IB.


( E ) HT29 cells were cultured overnight in serum free media and treated with vehicle or 50 ng / ml FGF19 for 30 min . The indicated proteins were detected by IB ( n = 3 culture plates ) .

O O O O O O O O O O O O O O O O O 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) HT29 cells were cultured overnight in serum free media and treated with vehicle or 50 ng/ml FGF19 for 30 min. The indicated proteins were detected by IB (n=3 culture plates).


( F ) FGF15 - KO mice or littermates were re - fed for 0 , 2 , or 3 h after fasting overnight . The indicated proteins in intestinal extracts were detected by IB ( n = 2 mice ) .

(F) FGF15-KO mice or littermates were re-fed for 0, 2, or 3 h after fasting overnight. The indicated proteins in intestinal extracts were detected by IB (n=2 mice).


( G ) HT29 cells were transfected with expression plasmids for flag - tagged TFEB or its mutants as indicated and 24 h later , cells were treated with FGF19 ( 50 ng / ml ) for 12 h . ∆ CT indicates the TFEB mutant with a C - terminal deletion of 15 amino acids . The indicated proteins were detected by IB ( n = 3 culture plates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were transfected with expression plasmids for flag-tagged TFEB or its mutants as indicated and 24 h later, cells were treated with FGF19 (50 ng/ml) for 12 h. ∆CT indicates the TFEB mutant with a C-terminal deletion of 15 amino acids. The indicated proteins were detected by IB (n=3 culture plates).


A - B Representative image of a proliferative cell in the DG labeled with an anti - PH3 antibody ( green ) ( A ) and quantification of the number of PH3 + cells in the DG of WT and Tau - / - mice ( B ) ( mean ± SEM ; n = 10 mice WT , n = 8 mice Tau - / - ; 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

A-B Representative image of a proliferative cell in the DG labeled with an anti-PH3 antibody (green) (A) and quantification of the number of PH3+ cells in the DG of WT and Tau-/- mice (B) (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).


C - D Representative image of an apoptotic cell in the DG labeled with an anti - fractin antibody ( red ) ( C ) and quantification of the number of fractin + cells in the DG of WT and Tau - / - mice ( D ) ( mean ± SEM ; n = 10 mice WT , n = 8 mice Tau - / - ; Student ´ s t - test ) .

C-D Representative image of an apoptotic cell in the DG labeled with an anti-fractin antibody (red) (C) and quantification of the number of fractin+ cells in the DG of WT and Tau-/- mice (D) (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).


E - F Schematic diagram of the cell survival experimental design using thymidine analogs ( F ) and quantification of the percentage of surviving cells in Tau - / - mice as compared to WT mice at each cell age ( 1 - , 2 - , 4 - , 6 - and 8 - week - old cells ) ( F ) ( mean ± SEM ( normalized data ) ; 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

E-F Schematic diagram of the cell survival experimental design using thymidine analogs (F) and quantification of the percentage of surviving cells in Tau-/- mice as compared to WT mice at each cell age (1-, 2-, 4-, 6- and 8-week-old cells) (F) (mean ± SEM (normalized data); Student´s t-test).


H Quantification of the percentage of 1 - , 4 - and 8 - week - old newborn neurons that express the DCX neuroblast marker and the NeuN mature neuron marker in WT and Tau - / - mice ( χ2 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

H Quantification of the percentage of 1-, 4- and 8-week-old newborn neurons that express the DCX neuroblast marker and the NeuN mature neuron marker in WT and Tau-/- mice (χ2 test).


I - J Representative images of progenitor cells in the DG labeled with anti - Sox2 ( red ) and anti - BLBP ( green ) antibodies ( I ) , and neuroblasts and immature neurons labeled with anti - DCX ( red ) and anti - calretinin ( green ) antibodies , respectively ( J ) . K - N Quantification of the number of Sox2 + ( K ) , BLBP + ( L ) , DCX + ( M ) and calretinin + ( N ) cells in the DG of WT and Tau - / - mice ( mean ± SEM ; n = 10 mice WT , n = 8 mice Tau - / - ; 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 O O O O O O O O O O O O O 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-J Representative images of progenitor cells in the DG labeled with anti-Sox2 (red) and anti-BLBP (green) antibodies (I), and neuroblasts and immature neurons labeled with anti-DCX (red) and anti-calretinin (green) antibodies, respectively (J).K-N Quantification of the number of Sox2+ (K), BLBP+ (L), DCX+ (M) and calretinin+ (N) cells in the DG of WT and Tau-/- mice (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).


A Representative images of 4 - week - old newborn granule neurons of WT and Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus . A schematic representation of Sholl ´ s analysis is shown in the WT image . B - C Quantification of total dendritic length ( B ) and Sholl ´ s analysis ( C ) of 4 - week - old newborn granule neurons in WT and Tau - / - mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of 4-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus. A schematic representation of Sholl´s analysis is shown in the WT image.B-C Quantification of total dendritic length (B) and Sholl´s analysis (C) of 4-week-old newborn granule neurons in WT and Tau-/- mice.


D Representative images of 8 - week - old newborn granule neurons of WT and Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus . E - F Quantification of total dendritic length ( E ) and Sholl ´ s analysis ( F ) of 8 - week - old newborn granule neurons in WT and Tau - / - mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of 8-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus.E-F Quantification of total dendritic length (E) and Sholl´s analysis (F) of 8-week-old newborn granule neurons in WT and Tau-/- mice.


G Representative images of 4 - week - old newborn granule neurons of WT and Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus and their corresponding high - power magnifications showing PSDs ( green ) . H - IQuantification of the number of PSDs / µm ( H ) and PSD area ( I ) in each dendritic branching order of 4 - week - old newborn granule neurons in WT and Tau - / - mice .

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G Representative images of 4-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green).H-IQuantification of the number of PSDs/µm (H) and PSD area (I) in each dendritic branching order of 4-week-old newborn granule neurons in WT and Tau-/- mice.


J Representative images of 8 - week - old newborn granule neurons of WT and Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus and their corresponding high - power magnifications showing PSDs ( green ) . K - L Quantification of the number of PSDs / µm ( K ) and PSD area ( L ) in each dendritic branching order of 8 - week - old newborn granule neurons in WT and Tau - / - mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of 8-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green).K-L Quantification of the number of PSDs/µm (K) and PSD area (L) in each dendritic branching order of 8-week-old newborn granule neurons in WT and Tau-/- mice.


A Representative traces of mEPSCs recorded at a holding potential of − 65 mV from WT ( black traces ) and Tau - / - ( red traces ) mice .

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A Representative traces of mEPSCs recorded at a holding potential of −65 mV from WT (black traces) and Tau-/- (red traces) mice.


B Exemplary traces of mEPSCs from one cell from WT ( black trace ) and Tau - / - ( red trace ) mice .

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B Exemplary traces of mEPSCs from one cell from WT (black trace) and Tau-/- (red trace) mice.


C Resting membrane potential of WT and Tau - / - mice ( mean ± SEM ; n = 13 cells WT , n = 10 cells Tau - / - ; * 0 . 05 > p ≥ 0 . 01 , Student ´ s t - test ) .

C Resting membrane potential of WT and Tau-/- mice (mean ± SEM; n= 13 cells WT, n= 10 cells Tau-/-; *0.05 > p ≥ 0.01, Student´s t-test).


D - E mEPSC amplitude ( D ) and mEPSC frequency ( E ) of WT and Tau - / - animals ( mean ± SEM ; n = 13 cells WT , n = 10 cells Tau - / - ; Student ´ s t test or Mann - Whitney ´ s U test ) .

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D-E mEPSC amplitude (D) and mEPSC frequency (E) of WT and Tau-/- animals (mean ± SEM; n= 13 cells WT, n= 10 cells Tau-/-; Student´s t test or Mann-Whitney´s U test).


F Cumulative probability plot of the total mEPSC amplitudes , small mEPSCs ( i ) and big mEPSCs ( ii ) from both WT and Tau - / - mice ( black and red traces , respectively ) ( * * * p < 0 . 001 ; Kolmogorov - Smirnov Z 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

F Cumulative probability plot of the total mEPSC amplitudes, small mEPSCs (i) and big mEPSCs (ii) from both WT and Tau-/- mice (black and red traces, respectively) (***p < 0.001; Kolmogorov-Smirnov Z test).


A Representative image of a mouse subjected to forced swimming in the Porsolt test .

A Representative image of a mouse subjected to forced swimming in the Porsolt test.


C Representative images of 1 - week - old newborn granule neurons labeled with CldU ( green ) belonging to WT CNP , WT P , Tau - / - CNP , and Tau - / - P mice . Cell nuclei were labeled with DAPI ( blue ) . Scale bar 50 µm . D - E Quantification of the number of 1 - week - old CldU + cells ( D ) and apoptotic fractin + cells ( E ) in each experimental group .

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 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

C Representative images of 1-week-old newborn granule neurons labeled with CldU (green) belonging to WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice. Cell nuclei were labeled with DAPI (blue). Scale bar 50 µm.D-E Quantification of the number of 1-week-old CldU+ cells (D) and apoptotic fractin+ cells (E) in each experimental group.


B Representative images of 8 - week - old newborn granule neurons of WT CNP , WT P , Tau - / - CNP , and Tau - / - P mice infected with a PSD95 - GFP - expressing retrovirus . In the WT CNP image a schematic representation of Sholl ´ s analysis is shown . C - D Quantification of total dendritic length ( C ) and Sholl ´ s analysis ( D ) of 8 - week - old newborn granule neurons ( n = 3 mice per experimental condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of 8-week-old newborn granule neurons of WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice infected with a PSD95-GFP-expressing retrovirus. In the WT CNP image a schematic representation of Sholl´s analysis is shown.C-D Quantification of total dendritic length (C) and Sholl´s analysis (D) of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).


E - F Representative images of an 8 - week - old newborn granule neuron , in which proximal dendrites are highlighted ( E ) , and of PSDs ( green ) in proximal dendrites belonging to WT CNP , WT P , Tau - / - CNP , and Tau - / - P mice ( F ) . G - H Quantification of the number of PSDs / µm ( G ) and PSD area ( H ) in each dendritic branching order of 8 - week - old newborn granule neurons ( n = 3 mice per experimental condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Representative images of an 8-week-old newborn granule neuron, in which proximal dendrites are highlighted (E), and of PSDs (green) in proximal dendrites belonging to WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice (F).G-H Quantification of the number of PSDs/µm (G) and PSD area (H) in each dendritic branching order of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).


I - J Representative Western Blot ( I ) and quantification ( J ) of the levels of GluR1 subunit of AMPA receptors in hippocampi of WT and Tau - / - mice subjected or not to the Porsolt test ( n = 6 mice per experimental condition ) .

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I-J Representative Western Blot (I) and quantification (J) of the levels of GluR1 subunit of AMPA receptors in hippocampi of WT and Tau-/- mice subjected or not to the Porsolt test (n= 6 mice per experimental condition).


K Representative image showing 8 - week - old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field ( indicated by a white square ) .

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K Representative image showing 8-week-old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field (indicated by a white square).


L - M Representative images ( L ) and quantification of the area ( M ) of MFTs of 8 - week - old newborn neurons in the CA3 region of WT CNP , WT P , Tau - / - CNP , and Tau - / - P mice ( n = 3 mice per experimental condition ) . Arrows indicate MFTs .

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

L-M Representative images (L) and quantification of the area (M) of MFTs of 8-week-old newborn neurons in the CA3 region of WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice (n= 3 mice per experimental condition). Arrows indicate MFTs.


A Representative images of control housing ( CH ) and environmental enrichment ( EE ) cages . B Schematic diagram of the experimental design .

A Representative images of control housing (CH) and environmental enrichment (EE) cages.B Schematic diagram of the experimental design.


C - D Representative images of DCX + cells in the DG of CH and EE animals ( C ) , and quantification of the number of DCX + cells of WT and Tau - / - animals in CH or EE conditions ( D ) .

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C-D Representative images of DCX+ cells in the DG of CH and EE animals (C), and quantification of the number of DCX+ cells of WT and Tau-/- animals in CH or EE conditions (D).


E Representative images of 8 - week - old newborn granule neurons labeled with IdU ( red ) belonging to WT CH , WT EE , Tau - / - CH and Tau - / - EE mice . F - G Quantification of the number of 8 - week - old IdU + cells ( F ) and apoptotic fractin + cells ( G ) .

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 O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

E Representative images of 8-week-old newborn granule neurons labeled with IdU (red) belonging to WT CH, WT EE, Tau-/- CH and Tau-/- EE mice.F-G Quantification of the number of 8-week-old IdU+ cells (F) and apoptotic fractin+ cells (G).


A Representative images of the whole hippocampus showing GABAergic innervation labeled with GAD - 65 antibody ( red ) , and high - power magnifications of the DG molecular layer showing GAD - 65 + terminals in WT CH , Tau - / - CH , WT EE and Tau - / - EE mice . Cell nuclei were labeled with DAPI ( blue ) . White scale bar 300 µm . Red scale bar 50 µm . Green scale bar 5 µm . B - C Quantification of the density ( B ) and area ( C ) of GAD - 65 + terminals in the molecular layer of mice in each experimental condition ( n = 10 mice WT CH , n = 8 miceTau - / - CH , n = 6 mice WT EE , n = 10 miceTau - / - EE ) .

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A Representative images of the whole hippocampus showing GABAergic innervation labeled with GAD-65 antibody (red), and high-power magnifications of the DG molecular layer showing GAD-65+ terminals in WT CH, Tau-/- CH, WT EE and Tau-/- EE mice. Cell nuclei were labeled with DAPI (blue). White scale bar 300 µm. Red scale bar 50 µm. Green scale bar 5 µm.B-C Quantification of the density (B) and area (C) of GAD-65+ terminals in the molecular layer of mice in each experimental condition (n= 10 mice WT CH, n= 8 miceTau-/- CH, n= 6 mice WT EE, n= 10 miceTau-/- EE).


B Representative images of 8 - week - old newborn granule neurons of WT CH , WT EE , Tau - / - CH , and Tau - / - EE mice infected with a PSD95 - GFP - expressing retrovirus . In the WT CH image a schematic representation of Sholl ´ s analysis is shown . C - D Quantification of total dendritic length ( C ) and Sholl ´ s analysis ( D ) of 8 - week - old newborn granule neurons ( n = 3 mice per experimental condition ) .

B Representative images of 8-week-old newborn granule neurons of WT CH, WT EE, Tau-/- CH, and Tau-/- EE mice infected with a PSD95-GFP-expressing retrovirus. In the WT CH image a schematic representation of Sholl´s analysis is shown.C-D Quantification of total dendritic length (C) and Sholl´s analysis (D) of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).


E - F Representative images of an 8 - week - old newborn granule neuron in which proximal and distal dendrites are highlighted ( E ) and of PSDs ( green ) in distal and proximal dendrites of WT and Tau - / - mice housed under either CH or EE conditions ( F ) . G - H Quantification of the number of PSDs / µm ( G ) and PSD area ( H ) in each dendritic branching order of 8 - week - old newborn granule neurons ( n = 3 mice per experimental condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Representative images of an 8-week-old newborn granule neuron in which proximal and distal dendrites are highlighted (E) and of PSDs (green) in distal and proximal dendrites of WT and Tau-/- mice housed under either CH or EE conditions (F).G-H Quantification of the number of PSDs/µm (G) and PSD area (H) in each dendritic branching order of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).


I Representative image showing 8 - week - old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field ( indicated by a white square ) .

I Representative image showing 8-week-old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field (indicated by a white square).


J - K Representative images ( J ) and quantification of the area ( K ) of MFTs of 8 - week - old newborn neurons in the CA3 region of WT CH , WT EE , Tau - / - CH , and Tau - / - EE mice ( n = 3 mice per experimental condition ) . Arrows indicate MFTs .

J-K Representative images (J) and quantification of the area (K) of MFTs of 8-week-old newborn neurons in the CA3 region of WT CH, WT EE, Tau-/- CH, and Tau-/- EE mice (n= 3 mice per experimental condition). Arrows indicate MFTs.


ChEP analysis using EGFP ( control ) - or Piwi - KD OSCs . Signals with an adjusted p - value ( Bonferroni test ) of less than 0 . 05 are highlighted in red . The signals for Lam , LamC , and H1 are labelled within the plots .

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ChEP analysis using EGFP (control)- or Piwi-KD OSCs. Signals with an adjusted p-value (Bonferroni test) of less than 0.05 are highlighted in red. The signals for Lam, LamC, and H1 are labelled within the plots.


Lam - or LamC - KD followed by qRT - PCR of RNA levels for piRNA target ( mdg1 and gypsy ) or non - target ( roo ) TEs . Error bars indicate SD ( n = 3 ) . * = p - value < 0 . 05 ( t - test ) . Note that the complete depletion of Lam and LamC is impossible since their KD causes severe damage to cells .

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Lam- or LamC-KD followed by qRT-PCR of RNA levels for piRNA target (mdg1 and gypsy) or non-target (roo) TEs. Error bars indicate SD (n=3). * = p-value<0.05 (t-test). Note that the complete depletion of Lam and LamC is impossible since their KD causes severe damage to cells.


Boxplots showing fold changes in the expression of piRNA target or non - target TEs based on RNA - seq upon Piwi - KD ( left ) , and the frequency of piRNAs , which target piRNA target TEs or non - piRNA target TEs ( right ) . Definition of piRNA target and non - target TEs is in Fig EV1E . Boxplot whiskers , box , and central band show maximum , third quartile , median , first quartile , and minimum , respectively ( n = 10 for piRNA target TEs , n = 80 for non - piRNA target TEs ) . P - values were calculated using the Wilcoxon rank - sum test .

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Boxplots showing fold changes in the expression of piRNA target or non-target TEs based on RNA-seq upon Piwi-KD (left), and the frequency of piRNAs, which target piRNA target TEs or non-piRNA target TEs (right). Definition of piRNA target and non-target TEs is in Fig EV1E. Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated using the Wilcoxon rank-sum test.


Boxplot showing DamID - seq signals of piRNA target or non - target TEs upon EGFP ( control ) - or Piwi - KD . Boxplot whiskers , box , and central band show maximum , third quartile , median , first quartile , and minimum , respectively ( n = 10 for piRNA target TEs , n = 80 for non - piRNA target TEs ) . P - values were calculated using the Wilcoxon rank - sum 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

Boxplot showing DamID-seq signals of piRNA target or non-target TEs upon EGFP (control)- or Piwi-KD. Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated using the Wilcoxon rank-sum test.


Oligo - FISH images for the representative piRNA target region described in Fig 4A , upon EGFP ( control ) - or Piwi - KD . Gray indicates DAPI staining for DNA , magenta indicates FISH signal , and green indicates Lamin immunofluorescence staining . Scale bar , 5μm . Ratio of distance from FISH signal to the nuclear periphery ( DAPI surface ) is quantified at right , dot in the box plot indicates mean value ; Boxplot whiskers , box , and central band show minimum , first quartile , median , third quartile , and maximum , respectively . P - values calculated with Mann - Whitney U 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 O O O O O O O O O O O O O O O O O O O O O

Oligo-FISH images for the representative piRNA target region described in Fig 4A, upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin immunofluorescence staining. Scale bar, 5μm. Ratio of distance from FISH signal to the nuclear periphery (DAPI surface) is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.


Oligo - FISH images for changes in the intra - TAD interaction at the TAD described in ( C ) , upon EGFP ( control ) - or Piwi - KD . Gray indicates DAPI staining for DNA , green and magenta indicates FISH signal at the different positions of TADs ( TAD - 1 and TAD - 2 ) . Details of TAD probe design is described in Fig EV2C . Scale bar , 5μm . Distance between two FISH signals is quantified at right , dot in the box plot indicates mean value ; Boxplot whiskers , box , and central band show minimum , first quartile , median , third quartile , and maximum , respectively . P - values calculated with Mann - Whitney U 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Oligo-FISH images for changes in the intra-TAD interaction at the TAD described in (C), upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, green and magenta indicates FISH signal at the different positions of TADs (TAD-1 and TAD-2). Details of TAD probe design is described in Fig EV2C. Scale bar, 5μm. Distance between two FISH signals is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.


Boxplots showing fold changes in the histone mark association of piRNA target or non - target TEs based on ChIP - seq upon Piwi - KD ( Definition of piRNA target and non - target TEs are described in Fig EV1E ) . Boxplot whiskers , box , and central band show maximum , third quartile , median , first quartile , and minimum , respectively ( n = 10 for piRNA target TEs , n = 80 for non - piRNA target TEs ) . P - values were calculated by Wilcoxon rank - sum test .

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Boxplots showing fold changes in the histone mark association of piRNA target or non-target TEs based on ChIP-seq upon Piwi-KD (Definition of piRNA target and non-target TEs are described in Fig EV1E). Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated by Wilcoxon rank-sum test.


ChIP ‐ qPCR analysis of H3K27Ac and H3K4me3 occupancy on the reporter gene upon transfection of λN - Nxf2 expression vector or control EGFP expression vector . Bar graph shows the occupancy relative to that of the sample transfected with a control plasmid . Error bars indicate SD ( n = 3 ) , * = p - value < 0 . 01 ( t - test ) . .

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ChIP‐qPCR analysis of H3K27Ac and H3K4me3 occupancy on the reporter gene upon transfection of λN-Nxf2 expression vector or control EGFP expression vector. Bar graph shows the occupancy relative to that of the sample transfected with a control plasmid. Error bars indicate SD (n = 3), * = p-value<0.01 (t-test)..


Immunoprecipitation ( IP ) from OSC lysate using anti - Lam , or myc antibody , followed by WB using indicated antibodies . Mouse immunoglobulin G ( n . i . ) was used for control IP . Note that OSCs used at the right panel is a line which stably expresses myc - Nxf2 .

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

Immunoprecipitation (IP) from OSC lysate using anti-Lam, or myc antibody, followed by WB using indicated antibodies. Mouse immunoglobulin G (n.i.) was used for control IP. Note that OSCs used at the right panel is a line which stably expresses myc-Nxf2.


Density plots for DamID - seq signals over consensus sequences from piRNA target ( gypsy and mdg1 , orange ) and non - target TEs ( roo , blue ) in EGFP ( control ) - or Nxf2 - KD cells .

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

Density plots for DamID-seq signals over consensus sequences from piRNA target (gypsy and mdg1, orange) and non-target TEs (roo, blue) in EGFP (control)- or Nxf2-KD cells.


Oligo - FISH images for the reporter of the tethering system , at 0h ( control ) , 48h hours , 96 hours after transfection of λN - Nxf2 expression vector . Gray indicates DAPI staining for DNA , magenta indicates FISH signal , and green indicates Lamin staining . Scale bar , 5μm . The ratio of distance from FISH signal to the nuclear periphery ( DAPI surface ) is quantified at right , dot in the box plot indicates mean value ; Boxplot whiskers , box , and central band show minimum , first quartile , median , third quartile , and maximum , respectively . P - values calculated with Mann - Whitney U test .

Oligo-FISH images for the reporter of the tethering system, at 0h (control), 48h hours, 96 hours after transfection of λN-Nxf2 expression vector. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin staining. Scale bar, 5μm. The ratio of distance from FISH signal to the nuclear periphery (DAPI surface) is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.


Dot plot showing the correlation between the normalized density of indicated TEs per TAD ( x - axis ) and changes in intra - TAD interactions upon H1 ( left ) - , Egg ( middle ) - , or HP1a ( right ) - KD ( y - axis ) . Trend lines are in red . Spearman ' s rank correlation rho is indicated along with the p - value ( algorithm AS 89 ) . n . s . ; not significant ( p > 0 . 05 ) Dot plot showing the correlation between the normalized density of indicated TEs per TAD ( x - axis ) and changes in inter - TAD interactions upon H1 ( left ) - , Egg ( middle ) - , or HP1a ( right ) - KD ( y - axis ) . Trend lines are in red . Spearman ' s rank correlation rho is indicated along with the p - value ( algorithm AS 89 ) . n . s . ; not significant ( p > 0 . 05 )

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Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in intra-TAD interactions upon H1(left)-, Egg(middle)-, or HP1a(right)-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). n.s.; not significant (p > 0.05) Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in inter-TAD interactions upon H1(left)-, Egg(middle)-, or HP1a(right)-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). n.s.; not significant (p > 0.05)


A , Western blotting showing cellular INSL5 in the immortalized NPEC and NPC cell lines after inhibition of protein secretion by BFA , and β - actin was used as a loading control .

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A, Western blotting showing cellular INSL5 in the immortalized NPEC and NPC cell lines after inhibition of protein secretion by BFA, and β-actin was used as a loading control.


B , ELISA assay showing secreted INSL5 in the supernatants of NPECs and NPC cell lines . For NPECs , n = 3 ; for NPC , n = 7 .

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B, ELISA assay showing secreted INSL5 in the supernatants of NPECs and NPC cell lines. For NPECs, n=3; for NPC, n=7.


C , The concentration of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - tumor individuals with VCA - IgA positive ( normal EBV ( + ) ) and NPC patients , in training cohort .

C, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in training cohort.


E , The concentration of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - tumor individuals with VCA - IgA positive ( normal EBV ( + ) ) and NPC patients , in validation cohort 1 ( GZ ) . F , The concentration of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - tumor individuals with VCA - IgA positive ( normal EBV ( + ) ) and NPC patients , in validation cohort 2 ( ZS ) .

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E, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 1(GZ). F, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 2(ZS).


A - C , Kaplan - Meier curves showing the overall survival ( OS ) curves of NPC patients with low or high INSL5 concentration ( A ) , low ( ≤ 4000 copy / ml ) or high ( > 4000 copy / ml ) EBV DNA copy number ( B ) , and the combination of INSL5 concentration with EBV DNA copy number ( C ) .

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A-C, Kaplan-Meier curves showing the overall survival (OS) curves of NPC patients with low or high INSL5 concentration (A), low (≤4000 copy/ml) or high (>4000 copy/ml) EBV DNA copy number (B), and the combination of INSL5 concentration with EBV DNA copy number (C).


D - F , Kaplan - Meier curves showing the disease - free survival ( DFS ) curves of NPC patients with low or high INSL5 concentration ( D ) , low or high EBV DNA copy number ( E ) , and the combination of INSL5 concentration with EBV DNA copy number ( F ) .

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D-F, Kaplan-Meier curves showing the disease-free survival (DFS) curves of NPC patients with low or high INSL5 concentration (D), low or high EBV DNA copy number (E), and the combination of INSL5 concentration with EBV DNA copy number (F).


A . Exogenous expression of INSL5 in NPC cells . Representative immunoblotting ( upper panel ) and immunofluorescent staining ( lower panel ) showed stable exogenous expression of INSL5 in both CNE1 , CNE2 and HK1 NPC cell lines . Scale bars represent 20μm .

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A. Exogenous expression of INSL5 in NPC cells. Representative immunoblotting (upper panel) and immunofluorescent staining (lower panel) showed stable exogenous expression of INSL5 in both CNE1, CNE2 and HK1 NPC cell lines. Scale bars represent 20μm.B


MTT assay of vector control or INSL5 overexpressing CNE1 , CNE2 and HK1 NPC cell lines ( upper panel ) either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 and # 2 ) ( lower panel ) . n = 4 biological replicates for CNE1 and CNE2 cell line , n = 6 biological replicates for HK1 .

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MTT assay of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines (upper panel) either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2) (lower panel). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1.


colony formation ( C and D ) of vector control or INSL5 overexpressing CNE1 , CNE2 and HK1 NPC cell lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 and # 2 ) . Representative images are shown in ( C ) for colony formation Number of colonies per field of view were plotted in D The results are from three different experiments .

colony formation (C and D) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (C) for colony formation Number of colonies per field of view were plotted in D The results are from three different experiments.


Brdu incorporation ( E and F ) of vector control or INSL5 overexpressing CNE1 , CNE2 and HK1 NPC cell lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 and # 2 ) . Representative images are shown in ( E ) Brdu incorporation the percentage of Brdu positive cells were plotted in The results are from three different experiments . Scale bars represent 20μm in ( E )

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Brdu incorporation (E and F) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (E) Brdu incorporation the percentage of Brdu positive cells were plotted in The results are from three different experiments. Scale bars represent 20μm in (E)


migration assays ( G and H ) of vector control or INSL5 overexpressing CNE1 , CNE2 and HK1 NPC cell lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 and # 2 ) . Representative images are shown in ( G ) for migration assays migrated cells per field of view were plotted in H , respectively . The results are from three different experiments . Scale bars represent 100μm in ( G )

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migration assays (G and H) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (G) for migration assays migrated cells per field of view were plotted in H, respectively. The results are from three different experiments. Scale bars represent 100μm in (G)


I , J , Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice . . Tumor size ( I ) n = 11 mice per group .

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I, J, Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor size (I) n=11 mice per group.


Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice . . Tumor weight of two groups . n = 11 mice per group .

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Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor weight of two groups. n=11 mice per group.


A , Gene expression profiles from CNE2 cells overexpressed with INSL5 or the vector control . The genes are shaded with green , black or red in the heat map to indicate low , intermediate or high expression , respectively .

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A, Gene expression profiles from CNE2 cells overexpressed with INSL5 or the vector control. The genes are shaded with green, black or red in the heat map to indicate low, intermediate or high expression, respectively.


C , The signal pathway of the subset of genes involved in metabolism was assessed by Gene Set Enrichment Analysis .

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C, The signal pathway of the subset of genes involved in metabolism was assessed by Gene Set Enrichment Analysis.


D , Analysis of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock - down cell lines .

D, Analysis of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock-down cell lines.


E , Metabolites were purified from five ( CNE1 cell line and HK1 cell line ) or seven ( CNE2 cell line ) independent samples of two vector and INSL5 overexpressed cell lines and analyzed by liquid chromatography - mass spectrometry ( LC - MS ) . Red : glycolytic intermediate metabolites . Green : tricarboxylic acid cycle ( TCA cycle ) intermediate metabolites . PPP : pentose phosphate pathway . The color scale bar represents Z - score of the metabolites ' level .

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E, Metabolites were purified from five (CNE1 cell line and HK1 cell line) or seven (CNE2 cell line) independent samples of two vector and INSL5 overexpressed cell lines and analyzed by liquid chromatography-mass spectrometry (LC-MS). Red: glycolytic intermediate metabolites. Green: tricarboxylic acid cycle (TCA cycle) intermediate metabolites. PPP: pentose phosphate pathway. The color scale bar represents Z-score of the metabolites' level.


F , The extracellular acidification rate ( ECAR ) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer . Glu , glucose ; Oligo , oligomycin .

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F, The extracellular acidification rate (ECAR) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer. Glu, glucose; Oligo, oligomycin.


G , Statistical analysis of the effects of INSL5 overexpression on glycolytic activity .

G, Statistical analysis of the effects of INSL5 overexpression on glycolytic activity.


H , CNE1 - vector and CNE1 - INSL5 cells were grown in the presence of the fluorescent glucose analog 2 - NBDG ( Invitrogen ) for 15min , and glucose uptake was then quantified using flow cytometry ( FACS ) ( left panel ) . Statistical analysis of the glucose uptake was showed in the right panel .

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H, CNE1-vector and CNE1-INSL5 cells were grown in the presence of the fluorescent glucose analog 2-NBDG (Invitrogen) for 15min, and glucose uptake was then quantified using flow cytometry (FACS) (left panel). Statistical analysis of the glucose uptake was showed in the right panel.


glucose consumption and lactate production ( I ) in CNE1 - vector and CNE1 - INSL5 cells .

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glucose consumption and lactate production (I) in CNE1-vector and CNE1-INSL5 cells.


basic ATP level or the ATP level of cell which was treated by 1uM oligomycin or 50uM 2 - DG for 24h ( J ) in CNE1 - vector and CNE1 - INSL5 cells .

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basic ATP level or the ATP level of cell which was treated by 1uM oligomycin or 50uM 2-DG for 24h (J) in CNE1-vector and CNE1-INSL5 cells.


HK2 enzyme activity ( K ) in CNE1 - vector and CNE1 - INSL5 cells .

HK2 enzyme activity (K) in CNE1-vector and CNE1-INSL5 cells.


L , ATP concentration , HK2 enzyme activity and lactate production in INSL5 wide - type or knock down HNE1 - EBV cells .

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L, ATP concentration, HK2 enzyme activity and lactate production in INSL5 wide-type or knock down HNE1-EBV cells.


B , phosphorylation of STAT5 under INSL5 stimulation . CNE1 cells were starved in FBS - free medium for 24h and then treated with INSL5 peptide ( 50ng / mL ) in the presence of an anti - INSL5 antibody at 100ug / ml , or an isotype - matched IgG control ( IgG ) for 30min .

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B, phosphorylation of STAT5 under INSL5 stimulation. CNE1 cells were starved in FBS-free medium for 24h and then treated with INSL5 peptide (50ng/mL) in the presence of an anti-INSL5 antibody at 100ug/ml, or an isotype-matched IgG control (IgG) for 30min.


C , Localization of STAT5 . A confocal analysis of STAT5 staining was performed using CNE1 cells transfected vector or INSL5 . Scale bars represent

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C, Localization of STAT5. A confocal analysis of STAT5 staining was performed using CNE1 cells transfected vector or INSL5. Scale bars represent 20μm


D , Cytoplasmic and nuclear proteins from vector or INSL5overexpressing cells were separated and subjected to immunoblot as indicated . The nuclear marker Lamin B1 and the cytoplasmic marker GAPDH were used to demonstrate the purity of fractions .

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D, Cytoplasmic and nuclear proteins from vector or INSL5overexpressing cells were separated and subjected to immunoblot as indicated. The nuclear marker Lamin B1 and the cytoplasmic marker GAPDH were used to demonstrate the purity of fractions.


E , INSL5 overexpression activated AKT , ERK , JAK1 and STAT5 phosphorylation , not STAT3 in HK1 cells . INSL5 overexpressing HK1 cells were treated with MK2206 ( 50uM ) , U0126 ( 20uM ) , MK2206 + U0126 , or Ruxolitinib ( 5uM ) for 24h . Western blotting was performed to evaluate the effects of those inhibitors on STAT5 and STAT3 phosphorylation level .

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E, INSL5 overexpression activated AKT, ERK, JAK1 and STAT5 phosphorylation, not STAT3 in HK1 cells. INSL5 overexpressing HK1 cells were treated with MK2206 (50uM), U0126(20uM), MK2206+U0126, or Ruxolitinib (5uM) for 24h. Western blotting was performed to evaluate the effects of those inhibitors on STAT5 and STAT3 phosphorylation level.


F , ChIP - qPCR analyzed the occupancy of potential E - boxes by STAT5 in the genes of HK2 , Glut3 and PFK1 in INSL5 - overexpressing CNE1 cells using IgG or anti - STAT5 antibodies .

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F, ChIP-qPCR analyzed the occupancy of potential E-boxes by STAT5 in the genes of HK2, Glut3 and PFK1 in INSL5-overexpressing CNE1 cells using IgG or anti-STAT5 antibodies.


G , Luciferase activity of different glycolytic gene promoter reporters in 293T cells transfected with STAT5 or empty vector .

G, Luciferase activity of different glycolytic gene promoter reporters in 293T cells transfected with STAT5 or empty vector.


H , Glucose uptake assessed by 2 - NBDG incubation in INSL5 overexpressed CNE1 cells with STAT5 knock down .

H, Glucose uptake assessed by 2-NBDG incubation in INSL5 overexpressed CNE1 cells with STAT5 knock down.


I , Glucose uptake was assessed by 2 - BNDG incubation in INSL5 overexpressed HK1 cells treated with JAK kinase inhibitor ( Ruxolitinib ) .

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I, Glucose uptake was assessed by 2-BNDG incubation in INSL5 overexpressed HK1 cells treated with JAK kinase inhibitor (Ruxolitinib).


J , MTT assay of vector control or INSL5 overexpressing HK1 cells either treated with STAT5 siRNAs or JAK kinase inhibitor .

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J, MTT assay of vector control or INSL5 overexpressing HK1 cells either treated with STAT5 siRNAs or JAK kinase inhibitor.


A , Brdu assay for CNE1 cells stimulated with 100ng / ml INSL5 peptide in the presence of an anti - INSL5 antibody at 50 or 100ug / ml , or an isotype - matched IgG control ( IgG ) for 24h . Scale bars represent 20μm .

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A, Brdu assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 20μm.


B , similar to ( A ) , migration and invasion assay for CNE1 cells stimulated with 100ng / ml INSL5 peptide in the presence of an anti - INSL5 antibody at 50 or 100ug / ml , or an isotype - matched IgG control ( IgG ) for 24h . Scale bars represent 100μm .

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B, similar to (A), migration and invasion assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 100μm.


C - E Growth curves of cell lines stably expressing INSL5 or vector 48 h post - treatment with the indicated dose of glycolysis inhibitor , 2 - DG . CNE1 cells ( C ) , CNE2 cells ( D ) and HK1 cells ( E ) . n = 4 biological replicates for CNE1 and CNE2 cell line , n = 6 biological replicates for

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C-E Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of glycolysis inhibitor, 2-DG. CNE1 cells (C), CNE2 cells (D) and HK1 cells (E). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1


F , Growth curves of cell lines stably expressing INSL5 or vector 48 h post - treatment with the indicated dose of cis - platinum ( DDP ) and 2 - DG . n = 4 biological replicates per group .

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F, Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of cis-platinum (DDP) and 2-DG. n=4 biological replicates per group.


Tumor size ( G ) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti - INSL5 , anti - GPCR142 or isotype - matched IgG control ( IgG ) every two days . n = 6 mice per group .

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Tumor size (G) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.


tumor weight ( H ) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti - INSL5 , anti - GPCR142 or isotype - matched IgG control ( IgG ) every two days . n = 6 mice per group .

tumor weight (H) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.


Representative confocal micrographs of immunostained Eph4 epithelial cell sheets in the apical plane . The apical MTs were associated with both LUZP1 and TJs . Scale bar , 10 μm ( low magnification ) and 1 μm ( high magnification ) . Representative immunoelectron micrographs of Eph4 cells . Immunogold particles for LUZP1 preferentially accumulated at the level of TJs that are positive for cingulin . MV , microvilli ; AJ , adherens junction , DS , desmosome . Scale bars , 200 nm .

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Representative confocal micrographs of immunostained Eph4 epithelial cell sheets in the apical plane. The apical MTs were associated with both LUZP1 and TJs. Scale bar, 10 μm (low magnification) and 1 μm (high magnification).   Representative immunoelectron micrographs of Eph4 cells. Immunogold particles for LUZP1 preferentially accumulated at the level of TJs that are positive for cingulin. MV, microvilli; AJ, adherens junction, DS, desmosome. Scale bars, 200 nm.


Representative confocal micrographs of co - cultures of wild - type ( WT ) and ZO - 1 / - 2 double knockout ( DKO ) Eph4 cells . ZO - 1 / - 2 DKO cells were marked by asterisks ( * ) . The LUZP1 junctional localization was apparently disrupted in TJ - deficient ZO - 1 / - 2 DKO cells . Scale bar , 10 μm . Representative confocal micrographs of transient E - cadherin knockout ( KO ) in WT Eph4 cells . E - cadherin KO cells were marked by asterisks ( * ) . The LUZP1 junctional localization seemed not to change between AJ - deficient E - cadherin KO and WT cells . Scale bar , 10 μm .

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Representative confocal micrographs of co-cultures of wild-type (WT) and ZO-1/-2 double knockout (DKO) Eph4 cells. ZO-1/-2 DKO cells were marked by asterisks (*). The LUZP1 junctional localization was apparently disrupted in TJ-deficient ZO-1/-2 DKO cells. Scale bar, 10 μm.   Representative confocal micrographs of transient E-cadherin knockout (KO) in WT Eph4 cells. E-cadherin KO cells were marked by asterisks (*). The LUZP1 junctional localization seemed not to change between AJ-deficient E-cadherin KO and WT cells. Scale bar, 10 μm.


Bar plots with dot density plots showing that ppMLC levels within CRs were significantly reduced in LUZP1 KO cells compared to those in WT cells [ 19 . 19 ± 10 . 02 arbitrary units ( a . u . ) ( WT ) vs . 2 . 92 ± 1 . 81 a . u . ( LUZP1 KO ) ] . n = 3 . * * p < 0 . 01 ( Mann - Whitney U test ) . Bars and error bars represent the mean ± SD .

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Bar plots with dot density plots showing that ppMLC levels within CRs were significantly reduced in LUZP1 KO cells compared to those in WT cells [19.19 ± 10.02 arbitrary units (a.u.) (WT) vs. 2.92 ± 1.81 a.u. (LUZP1 KO)]. n = 3. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± SD.


Representative confocal micrographs of co - cultures of wild - type ( WT ) and ZO - 1 / - 2 double knockout ( DKO ) Eph4 cells . ppMLC levels within CRs were clearly reduced in ZO - 1 / - 2 DKO cells compared to those in WT cells . Scale bar , 10 μm . Bar plots with dot density plots showing that apical areas of WT cells were significantly smaller than those of ZO - 1 / - 2 DKO cells [ 119 . 62 ± 31 . 62 μm2 ( WT ) vs . 199 . 96 ± 94 . 22 μm2 ( LUZP1 KO ) ] ( See also Fig 1I ) . n = 3 . * * p < 0 . 01 ( unpaired t test ) . Bars and error bars represent the mean ± SD .

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Representative confocal micrographs of co-cultures of wild-type (WT) and ZO-1/-2 double knockout (DKO) Eph4 cells. ppMLC levels within CRs were clearly reduced in ZO-1/-2 DKO cells compared to those in WT cells. Scale bar, 10 μm.   Bar plots with dot density plots showing that apical areas of WT cells were significantly smaller than those of ZO-1/-2 DKO cells [119.62 ± 31.62 μm2 (WT) vs. 199.96 ± 94.22 μm2 (LUZP1 KO)] (See also Fig 1I). n = 3. **p < 0.01 (unpaired t test). Bars and error bars represent the mean ± SD.


In vitro direct binding assay between FLAG - LUZP1 and GST - MLC . Representative immunoblots are shown . S , supernatant ; P , pellet . A representative plot in which the amount of bound GST - WT - , AA - , or DD - MLC / FLAG - LUZP1 was plotted with the Michaelis - Menten fitted curve . Bar plots with dot density plots showing that the dissociation constant ( Kd ) of LUZP1 from DD - MLC was significantly lower than that from WT - MLC and AA - MLC [ 4 . 04 ± 1 . 05 μM ( WT - MLC ) vs . 4 . 88 ± 1 . 79 μM ( AA - MLC ) vs . 0 . 85 ± 0 . 39 μM ( DD - MLC ) ] . n = 5 ( WT - MLC and AA - MLC ) and n = 4 ( DD - MLC ) . * p < 0 . 05 [ Kruskal - Wallis test followed by Steel - Dwass test ] . Bars and error bars represent the mean ± SD .

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

In vitro direct binding assay between FLAG-LUZP1 and GST-MLC. Representative immunoblots are shown. S, supernatant; P, pellet.   A representative plot in which the amount of bound GST-WT-, AA-, or DD-MLC/FLAG-LUZP1 was plotted with the Michaelis-Menten fitted curve.   Bar plots with dot density plots showing that the dissociation constant (Kd) of LUZP1 from DD-MLC was significantly lower than that from WT-MLC and AA-MLC [4.04 ± 1.05 μM (WT-MLC) vs. 4.88 ± 1.79 μM (AA-MLC) vs. 0.85 ± 0.39 μM (DD-MLC)]. n = 5 (WT-MLC and AA-MLC) and n = 4 (DD-MLC). *p < 0.05 [Kruskal-Wallis test followed by Steel-Dwass test]. Bars and error bars represent the mean ± SD.


Representative immunoblot of WT , LUZP1 KO , and Venus - LUZP1 - expressing LUZP1 knockout ( REV ) Eph4 cells treated with 100 nM calyculin A for 30 min . Quantification of the ppMLC / MLC ratio relative to WT control , confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A [ WT , 1 . 00 ( control ) vs . 1 . 40 ± 0 . 06 ( calyculin A ) vs . 1 . 14 ± 0 . 33 ( washout ) ; KO , 0 . 09 ± 0 . 04 ( control ) vs . 1 . 49 ± 0 . 06 ( calyculin A ) vs . 0 . 81 ± 0 . 99 ( washout ) ; REV , 2 . 06 ± 1 . 78 ( control ) vs . 1 . 82 ± 1 . 50 ( calyculin A ) vs . 1 . 80 ± 1 . 14 ( washout ) ] . n = 3 . Bars and error bars represent the mean ± SD .

O 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 O O O O O O O O O O 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 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 O O O O O O O O O O O O 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 O O O O O O O O O O O O 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 O O O O O 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 immunoblot of WT, LUZP1 KO, and Venus-LUZP1-expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A [WT, 1.00 (control) vs. 1.40 ± 0.06 (calyculin A) vs. 1.14 ± 0.33 (washout); KO, 0.09 ± 0.04 (control) vs. 1.49 ± 0.06 (calyculin A) vs. 0.81 ± 0.99 (washout); REV, 2.06 ± 1.78 (control) vs. 1.82 ± 1.50 (calyculin A) vs. 1.80 ± 1.14 (washout)]. n = 3. Bars and error bars represent the mean ± SD.


In vitro MLC phosphorylation assay using 1 μg GST - PP1c β / δ in addition to 25 ng GST - MLC , 4 ng GST - ROCK1 catalytic domain , 1 mM ATP , and 0 - 5 μg GST - LUZP1 . Quantification of the di - phosphorylated MLC ( ppMLC ) / MLC ratio relative to the control showed that LUZP1 up - regulated ppMLC / MLC levels in a dose - dependent manner [ 1 . 00 ( 1st lane , control ) vs . 1 . 27 ± 0 . 33 ( 2nd lane ) vs . 1 . 76 ± 0 . 68 ( 3rd lane ) vs . 2 . 53 ± 1 . 65 ( 4th lane ) vs . 2 . 93 ± 2 . 45 ( 5th lane ) ] . n = 3 or 6 . * * p < 0 . 01 [ Kruskal - Wallis test followed by Steel test ( compared with 1st lane ) ] . Bars and error bars represent the mean ± SD . In vitro Merlin phosphorylation assay using 1 μg GST - PP1c β / δ , 100 ng GST - Merlin , 2 pg p21 - activated kinase 1 ( PAK1 ) , and 5 μg GST - LUZP1 . Quantification of the phosphorylated Merlin ( pMerlin ) / Merlin ratio relative to the control showed that LUZP1 up - regulated pMerlin / Merlin levels [ 0 . 23 ± 0 . 15 ( 1st lane ) vs . 1 . 00 ( 2nd lane , control ) vs . 0 . 32 ± 0 . 17 ( 3rd lane ) vs . 0 . 97 ± 0 . 42 ( 4th lane ) vs . 1 . 25 ± 0 . 39 ( 5th lane ) ] . n = 4 or 9 . * p < 0 . 05 , * * p < 0 . 01 [ Kruskal - Wallis test followed by Steel test ( compared with 3rd lane ) ] . Bars and error bars represent the mean ± SD .

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

In vitro MLC phosphorylation assay using 1 μg GST-PP1c β/δ in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, and 0-5 μg GST-LUZP1. Quantification of the di-phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 up-regulated ppMLC/MLC levels in a dose-dependent manner [1.00 (1st lane, control) vs. 1.27 ± 0.33 (2nd lane) vs. 1.76 ± 0.68 (3rd lane) vs. 2.53 ± 1.65 (4th lane) vs. 2.93 ± 2.45 (5th lane)]. n = 3 or 6. **p < 0.01 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD.   In vitro Merlin phosphorylation assay using 1 μg GST-PP1c β/δ, 100 ng GST-Merlin, 2 pg p21-activated kinase 1 (PAK1), and 5 μg GST-LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 up-regulated pMerlin/Merlin levels [0.23 ± 0.15 (1st lane) vs. 1.00 (2nd lane, control) vs. 0.32 ± 0.17 (3rd lane) vs. 0.97 ± 0.42 (4th lane) vs. 1.25 ± 0.39 (5th lane)]. n = 4 or 9. *p < 0.05, **p < 0.01 [Kruskal-Wallis test followed by Steel test (compared with 3rd lane)]. Bars and error bars represent the mean ± SD.


Box plots with dot density plots showing the ratio of the apical area / basal area in co - cultures of Venus - LUZP1 - expressing LUZP1 knockout ( REV ) and LUZP1 knockout ( LUZP1 KO ) Eph4 cells ; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells [ REV , 0 . 65 ± 0 . 16 ( control ) vs . 0 . 90 ± 0 . 18 ( nocodazole ) vs . 0 . 64 ± 0 . 16 ( washout ) ; KO , 1 . 30 ± 0 . 17 ( control ) vs . 1 . 07 ± 0 . 13 ( nocodazole ) vs . 1 . 32 ± 0 . 19 ( washout ) ] . * * p < 0 . 01 ( Kruskal - Wallis test followed by Steel - Dwass test ) . The solid lines represent the medians and the boxes represent the inter - quartile ranges . The error bars extending from the box represents the data within 1 . 5 times of the interquartile range . Representative confocal micrographs of co - cultures of LUZP1 - expressing wild - type ( WT ) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min . Nocodazole treatment partially reversed the difference in di - phosphorylated MLC ( ppMLC ) levels within circumferential rings ( CRs ) between WT and LUZP1 KO cells . Scale bar , 10 μm . Bar plots with dot density plots showing that ppMLC levels within CRs were significantly down - regulated in WT Eph4 cells after nocodazole treatment . Importantly , ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment [ WT , 21 . 43 ± 6 . 96 arbitrary units ( a . u . ) ( control ) vs . 17 . 67 ± 5 . 40 a . u . ( nocodazole ) vs . 20 . 84 ± 7 . 19 a . u . ( washout ) ; KO , 8 . 74 ± 1 . 71 a . u . ( control ) vs . 8 . 67 ± 1 . 89 a . u . ( nocodazole ) vs . 7 . 96 ± 2 . 35 a . u . ( washout ) ] . n = 3 . * * p < 0 . 01 ( Kruskal - Wallis test followed by Steel - Dwass test ) . Bars and error bars represent the mean ± standard deviation ( SD ) .

O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O 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 O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-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 O O O O O O 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 O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Box plots with dot density plots showing the ratio of the apical area/basal area in co-cultures of Venus-LUZP1-expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells [REV, 0.65 ± 0.16 (control) vs. 0.90 ± 0.18 (nocodazole) vs. 0.64 ± 0.16 (washout); KO, 1.30 ± 0.17 (control) vs. 1.07 ± 0.13 (nocodazole) vs. 1.32 ± 0.19 (washout)]. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). The solid lines represent the medians and the boxes represent the inter-quartile ranges. The error bars extending from the box represents the data within 1.5 times of the interquartile range. Representative confocal micrographs of co-cultures of LUZP1-expressing wild-type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di-phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm.   Bar plots with dot density plots showing that ppMLC levels within CRs were significantly down-regulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment [WT, 21.43 ± 6.96 arbitrary units (a.u.) (control) vs. 17.67 ± 5.40 a.u. (nocodazole) vs. 20.84 ± 7.19 a.u. (washout); KO, 8.74 ± 1.71 a.u. (control) vs. 8.67 ± 1.89 a.u. (nocodazole) vs. 7.96 ± 2.35 a.u. (washout)]. n = 3. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). Bars and error bars represent the mean ± standard deviation (SD).


In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST - MLC , 4 ng GST - ROCK1 catalytic domain , 1 mM ATP , 1 μg GST - protein phosphatase 1c β / δ ( PP1c β / δ ) , and 0 - 5 μg GST - LUZP1 . Quantification of the relative ppMLC / MLC ratio to the control showed that MTs promote LUZP1 - mediated inhibition of PP1c β / δ [ 1 . 00 ( 1st - lane , control ) vs . 1 . 42 ± 0 . 59 ( 2nd - lane ) vs . 1 . 72 ± 0 . 76 ( 3rd - lane ) vs . 1 . 99 ± 0 . 56 ( 4th - lane ) vs . 1 . 14 ± 0 . 37 ( 5th - lane ) vs . 2 . 87 ± 1 . 51 ( 6th - lane ) vs . 2 . 74 ± 1 . 19 ( 7th - lane ) vs . 2 . 50 ± 0 . 88 ( 8th - lane ) ] . n = 6 . * p < 0 . 05 [ Kruskal - Wallis test followed by Steel test ( compared with 1st lane ) ] . Bars and error bars represent the mean ± SD .

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

In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, 1 μg GST-protein phosphatase 1c β/δ (PP1c β/δ), and 0-5 μg GST-LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1-mediated inhibition of PP1c β/δ [1.00 (1st-lane, control) vs. 1.42 ± 0.59 (2nd-lane) vs. 1.72 ± 0.76 (3rd-lane) vs. 1.99 ± 0.56 (4th-lane) vs. 1.14 ± 0.37 (5th-lane) vs. 2.87 ± 1.51 (6th-lane) vs. 2.74 ± 1.19 (7th-lane) vs. 2.50 ± 0.88 (8th-lane)]. n = 6. *p < 0.05 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD.


A , B Tumor growth curve ( left ) and summary of end - point tumor masses ( right ) of 6 - 8 week - old wildtype ( WT ) and Peli1 - KO ( KO ) mice inoculated s . c . with B16F10 ( A ; WT , n = 7 ; KO , n = 6 ) or E . G7 ( B ; WT , n = 5 ; KO , n = 5 ) tumor cells .

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

A,B Tumor growth curve (left) and summary of end-point tumor masses (right) of 6-8 week-old wildtype (WT) and Peli1-KO (KO) mice inoculated s.c. with B16F10 (A; WT, n=7; KO, n=6) or E.G7 (B; WT, n=5; KO, n=5) tumor cells.


C , D Flow cytometry analysis of CD4 + and CD8 + T cells in CD45 . 2 + TILs and dLN cells from B16F10 tumor - bearing wildtype and Peli1 - KO mice , presented as a representative plot ( C ) and summary graph based on multiple mice ( D ; WT , n = 6 ; KO , n = 6 ) .

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

C,D Flow cytometry analysis of CD4+ and CD8+ T cells in CD45.2+ TILs and dLN cells from B16F10 tumor-bearing wildtype and Peli1-KO mice, presented as a representative plot (C) and summary graph based on multiple mice (D; WT, n=6; KO, n=6).


E , F Flow cytometry analysis of IFNγ - producing CD4 + and CD8 + T cells in TILs , and dLN cells of B16F10 tumor - bearing ( day 19 ) wildtype and Peli1 - KO mice , presented as a representative plot ( E ) and summary graph ( F ) . ( WT , n = 6 ; KO , n = 6 )

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

E,F Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs, and dLN cells of B16F10 tumor-bearing (day 19) wildtype and Peli1-KO mice, presented as a representative plot (E) and summary graph (F). (WT, n=6; KO, n=6)


G , H Flow cytometry analysis of IFNγ + CD8 + T cells in TILs of E . G7 tumor - bearing ( day 24 ) wildtype and Peli1 - KO mice , presented as a representative plot ( G ) and summary graph ( H ) . ( WT , n = 3 ; KO , n = 3 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,H Flow cytometry analysis of IFNγ+CD8+ T cells in TILs of E.G7 tumor-bearing (day 24) wildtype and Peli1-KO mice, presented as a representative plot (G) and summary graph (H). (WT, n=3; KO, n=3)


A , B Tumor growth curve ( left ) and summary graph of end - point tumor masses ( right ) of wildtype ( WT ) and Peli1 - TKO ( TKO ) mice inoculated s . c . with B16 - OVA ( A ; WT , n = 5 ; KO , n = 5 ) or E . G7 ( B ; WT , n = 6 ; KO , n = 4 ) tumor cells .

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

A,B Tumor growth curve (left) and summary graph of end-point tumor masses (right) of wildtype (WT) and Peli1-TKO (TKO) mice inoculated s.c. with B16-OVA (A; WT, n=5; KO, n=5) or E.G7 (B; WT, n=6; KO, n=4) tumor cells.


C Flow cytometry analysis of IFNγ - producing CD4 + and CD8 + T cells in TILs and dLN cells from B16OVA tumor - bearing wildtype and Peli1 - TKO mice , presented as a representative plot ( upper ) and summary graph ( lower ) . ( WT , n = 5 ; KO , n = 5 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs and dLN cells from B16OVA tumor-bearing wildtype and Peli1-TKO mice, presented as a representative plot (upper) and summary graph (lower). (WT, n=5; KO, n=5)


D Flow cytometry analysis of GZMB + CD8 + T cells in TILs from B16 - OVA tumor - bearing wildtype and Peli1 - TKO mice , presented as a representative plot ( upper ) and summary graph ( lower ) . ( WT , n = 5 ; KO , n = 5 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Flow cytometry analysis of GZMB+CD8+ T cells in TILs from B16-OVA tumor-bearing wildtype and Peli1-TKO mice, presented as a representative plot (upper) and summary graph (lower). (WT, n=5; KO, n=5)


E Tumor growth curve ( left ) and summary graph of end - point tumor masses ( right ) of B16 - OVA tumor - bearing wildtype and the indicated Peli1 conditional KO mice . ( Treg KO model : WT , n = 5 ; Treg KO , n = 6 ; MKO model : WT , n = 3 ; MKO , n = 5 ; BKO model : WT , n = 9 ; BKO , n = 9 )

E Tumor growth curve (left) and summary graph of end-point tumor masses (right) of B16-OVA tumor-bearing wildtype and the indicated Peli1 conditional KO mice. (Treg KO model: WT, n=5; Treg KO, n=6; MKO model: WT, n=3; MKO, n=5; BKO model: WT, n=9; BKO, n=9)


A , B Seahorse analysis of basal ( glucose injection ) and maximal ( oligomycin injection ) ECAR or basal ( no treatment ) and maximal ( FCCP injection ) OCR in WT and Peli1 - KO OT - I CD8 T cells that were either untreated ( naïve ) or activated with plate - bound anti - CD3 ( 1 μg / ml ) plus anti - CD28 ( 1 μg / ml ) for 16 h . Data are presented as a representative plot ( A ) or summary graph based on 6 wildtype and 6 KO mice ( B ) .

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

A,B Seahorse analysis of basal (glucose injection) and maximal (oligomycin injection) ECAR or basal (no treatment) and maximal (FCCP injection) OCR in WT and Peli1-KO OT-I CD8 T cells that were either untreated (naïve) or activated with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) for 16 h. Data are presented as a representative plot (A) or summary graph based on 6 wildtype and 6 KO mice (B).


qRT - PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1 - KO OT - I CD8 T cells stimulated with anti - CD3 and anti - CD28 for 6 h .

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

qRT-PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1-KO OT-I CD8 T cells stimulated with anti-CD3 and anti-CD28 for 6 h.


D qRT - PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1 - KO OT - I CD8 T cells stimulated with anti - CD3 and anti - CD28 for 6 h .

O O O O O O O O O O O O O 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 qRT-PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1-KO OT-I CD8 T cells stimulated with anti-CD3 and anti-CD28 for 6 h.


E Immunoblot analysis of HK2 and Glut1 in wildtype or Peli1 - KO OT - I CD8 T cells that were stimulated with plate - bound anti - CD3 and anti - CD28 for 24 h .

O O O O O O O O O O O O O O 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 analysis of HK2 and Glut1 in wildtype or Peli1-KO OT-I CD8 T cells that were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 h.


A Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of freshly isolated CD8 T cells from wildtype ( WT ) or Peli1 KO ( KO ) mice ( 6 weeks old ) . Data are presented as a representative blot ( left ) and summary graphs of densitometric quantifications of the indicated proteins ( presented as phosphorylated / total protein ratio ) based on three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of freshly isolated CD8 T cells from wildtype (WT) or Peli1 KO (KO) mice (6 weeks old). Data are presented as a representative blot (left) and summary graphs of densitometric quantifications of the indicated proteins (presented as phosphorylated/total protein ratio) based on three independent experiments.


B , C Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype or Peli1 - KO OT - I CD8 T cells that were first rested on ice for 15 min and then stimulated in low - serum ( 1 % FBS ) medium with anti - CD3 and anti - CD28 for the indicated time periods .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,C Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were first rested on ice for 15 min and then stimulated in low-serum (1% FBS) medium with anti-CD3 and anti-CD28 for the indicated time periods.