C An example of neurons stained with the ROS sensible fluorescent probe DCF and the nuclei dye Hoechst . Anti - NCAM antibody was used to detect neurons . Scale bar 20 μm . Plots of the DCF fluorescence signal from NCAM + controls - and PKAN humanneurons differentiated or not in the presence of CoA ( 25 μM ) in the medium for 3 weeks . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

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 O O 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 An example of neurons stained with the ROS sensible fluorescent probe DCF and the nuclei dye Hoechst. Anti-NCAM antibody was used to detect neurons. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM+ controls- and PKAN humanneurons differentiated or not in the presence of CoA (25 μM) in the medium for 3 weeks. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).


D Oxygen consumption rate ( OCR ) with and without CoA on controls and PKAN patients . Basal and uncoupled ( FCCP ) respiration increased after CoA supplementation . Data presented as means + SEM of 24 independent replicates for each condition ( Two - way ANOVA ) .

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

D Oxygen consumption rate (OCR) with and without CoA on controls and PKAN patients. Basal and uncoupled (FCCP) respiration increased after CoA supplementation. Data presented as means + SEM of 24 independent replicates for each condition (Two-way ANOVA).


E Heme quantification by absorbance at 400 nm of the soluble NPC cell lysates . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

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

E Heme quantification by absorbance at 400 nm of the soluble NPC cell lysates. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).


( A , B ) Immunoblot analysis of the phosphorylation level of CLIP - 170 , AMPK and ACC in heart ( A ) and liver ( B ) along with the developmental stages . α - tubulin was used as a loading control . ( C - E ) Quantitative analysis of western blotting shown in Fig 1A . ( F - H ) Quantitative analysis of western blotting shown in Fig 1B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of the phosphorylation level of CLIP-170, AMPK and ACC in heart (A) and liver (B) along with the developmental stages. α-tubulin was used as a loading control. (C-E) Quantitative analysis of western blotting shown in Fig 1A. (F-H) Quantitative analysis of western blotting shown in Fig 1B.


( I ) Immunostained images of adult mouse heart tissue . These were stained with a phosphorylated AMPK antibody ( I , upper ) , a phosphorylated CLIP - 170 antibody ( I , lower ) and a plakoglobin antibody ( middle ) . The representative images from more than 5 analyzed tissues were shown .

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

(I) Immunostained images of adult mouse heart tissue. These were stained with a phosphorylated AMPK antibody (I, upper), a phosphorylated CLIP-170 antibody (I, lower) and a plakoglobin antibody (middle). The representative images from more than 5 analyzed tissues were shown.


( A , B ) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0 . 01 % DMSO ( Control , upper row ) or 2 μM MYK - 461 ( MYK - 461 , middle row ) and 4 hours after washing out of MYK - 461 ( Wash out , bottom row ) . These cells were stained with a phosphorylated AMPK antibody , an AMPKβ2 antibody and a N - cadherin antibody .

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

(A, B) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, middle row) and 4 hours after washing out of MYK-461 (Wash out, bottom row). These cells were stained with a phosphorylated AMPK antibody, an AMPKβ2 antibody and a N-cadherin antibody.


( C , D ) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0 . 01 % DMSO ( Control , upper row ) or 2 μM MYK - 461 ( MYK - 461 , bottom row ) and stained with a connexin43 antibody , a plakoglobin antibody , an LKB - 1 antibody and an N - cadherin antibody . The representative images from 4 independent experiments were shown .

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

(C, D) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, bottom row) and stained with a connexin43 antibody, a plakoglobin antibody, an LKB-1 antibody and an N-cadherin antibody. The representative images from 4 independent experiments were shown.


( E ) Quantification of pAMPK , AMPKβ2 , Connexin43 or LKB1 that was localized with a cell - cell junction marker ( N - cadherin or Plakoglobin ) . Box and whisker plots showed the 25th percentile ( bottom line of each box ) , median ( middle line of each box ) , 75th percentile ( top line of each box ) , maximum and minimum ( each whisker ) . Numbers in the brackets indicate the cells that were analyzed for quantification . Differences among multiple groups were compared by one - way ANOVA , followed by a post hoc comparison using the Tukey method . Two - tailed Student ' s t - test was used to analyze differences between two groups . * , P < 0 . 05 * * , P < 0 . 01 . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of pAMPK, AMPKβ2, Connexin43 or LKB1 that was localized with a cell-cell junction marker (N-cadherin or Plakoglobin). Box and whisker plots showed the 25th percentile (bottom line of each box), median (middle line of each box), 75th percentile (top line of each box), maximum and minimum (each whisker). Numbers in the brackets indicate the cells that were analyzed for quantification. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. Two-tailed Student's t-test was used to analyze differences between two groups. *, P<0.05 **, P<0.01. n.s., not significant.


( A ) GFP time - lapse images of neonatal rat cardiomyocytes expressing EGFP - CLIP - 170 WT 0 min and 15 min after treatment with 20 μM Compound C ( left side panel ) and expressing EGFP - CLIP - 170 S311A ( right side panel ) . Higher magniﬁcation of white square ( upper row ) showing CLIP - 170 migrated longitudinally toward the cell - cell junctions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT 0 min and 15 min after treatment with 20 μM Compound C (left side panel) and expressing EGFP-CLIP-170 S311A (right side panel). Higher magniﬁcation of white square (upper row) showing CLIP-170 migrated longitudinally toward the cell-cell junctions.


( B ) Beeswarm plots of comet length of EGFP - CLIP - 170 . The comet length from multiple cells in different fields was analyzed . Number of comets analyzed , Pre : n = 32 , Cpd . C : n = 36 , CLIP S311A : n = 68 . Data means ± S . D . Differences among multiple groups were compared by one - way ANOVA , followed by a post hoc comparison using the Tukey method . * * , P < 0 . 01 versus Pre .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Beeswarm plots of comet length of EGFP-CLIP-170. The comet length from multiple cells in different fields was analyzed. Number of comets analyzed, Pre: n=32, Cpd. C: n=36, CLIP S311A: n=68. Data means ±S.D. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. **, P<0.01 versus Pre.


( C ) GFP time - lapse images of neonatal rat cardiomyocytes expressing EGFP - CLIP - 170 WT treated with control siRNA ( siCL , left ) or siRNA targeting both AMPKα1 and α2 ( siAMPKα1α2 , right ) . White dotted lines in the images showed the connected cardiomyocyte not expressing EGFP - CLIP - 170 WT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT treated with control siRNA (siCL, left) or siRNA targeting both AMPKα1 and α2 (siAMPKα1α2, right). White dotted lines in the images showed the connected cardiomyocyte not expressing EGFP-CLIP-170 WT.


( D ) Beeswarm plots of comet length of EGFP - CLIP - 170 . The comet length from multiple cells from different fields was analyzed . Number of comets analyzed , siCL : n = 178 , siAMPKα1α2 : n = 184 . Data means ± S . D . Two - tailed Student ' s t - test was used to analyze differences between two groups . * * , P < 0 . 01 versus siCL .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Beeswarm plots of comet length of EGFP-CLIP-170. The comet length from multiple cells from different fields was analyzed. Number of comets analyzed, siCL: n=178, siAMPKα1α2: n=184. Data means ±S.D. Two-tailed Student's t-test was used to analyze differences between two groups. **, P<0.01 versus siCL.


( A ) GFP time - lapse images of neonatal rat cardiomyocytes expressing EGFP - CLIP - 170 WT 0 , 2 and 3 hours after treatment with 2 μM MYK - 461 .

O O O O O O 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

(A) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT 0, 2 and 3 hours after treatment with 2 μM MYK-461.


( B ) Immunostained images of neonatal rat cardiomyocytes 4 hours after treatment with 0 . 01 % DMSO ( Control , upper row ) or 2 μM MYK - 461 ( MYK - 461 , bottom row ) . These cells were stained with an α - tubulin ( green ) and a plakoglobin ( red ) antibody .

O O O O 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 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

(B) Immunostained images of neonatal rat cardiomyocytes 4 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, bottom row). These cells were stained with an α-tubulin (green) and a plakoglobin (red) antibody.


( C ) Box and whisker plots of the cell size of a cardiomyocyte 2 hours after treatment with or without 2 μM MYK - 461 , showing the 25th percentile ( bottom line of each box ) , median ( middle line of each box ) , 75th percentile ( top line of each box ) , maximum and minimum ( each whisker ) . Adenovirus expressing EGFP - CLIP - 170 ( adCLIP ) mutant was used ( WT , S311A , S311D ) . Numbers in the graph indicate n number of the cells from 2 independent experiments . Control ( - / - ) : n = 784 , MYK - 461 : n = 939 , CLIP WT : n = 656 , CLIP WT + MYK - 461 : n = 619 , CLIP S311A : n = 405 , CLIP S311A + MYK - 461 : n = 635 , CLIP S311D : n = 613 , CLIP S311D + MYK - 461 : n = 735 . Data means ± S . D . Differences among multiple groups were compared by one - way ANOVA , followed by a post hoc comparison using the Tukey method . * * , P < 0 . 01 versus Control , † † , P < 0 . 01 versus CLIP WT , n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Box and whisker plots of the cell size of a cardiomyocyte 2 hours after treatment with or without 2 μM MYK-461, showing the 25th percentile (bottom line of each box), median (middle line of each box), 75th percentile (top line of each box), maximum and minimum (each whisker). Adenovirus expressing EGFP-CLIP-170 (adCLIP) mutant was used (WT, S311A, S311D). Numbers in the graph indicate n number of the cells from 2 independent experiments. Control (-/-): n=784, MYK-461: n=939, CLIP WT: n=656, CLIP WT + MYK-461: n=619, CLIP S311A: n=405, CLIP S311A + MYK-461: n=635, CLIP S311D: n=613, CLIP S311D + MYK-461: n=735. Data means ±S.D. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. **, P<0.01 versus Control, ††, P<0.01 versus CLIP WT, n.s., not significant.


( A ) Beeswarm plots of the echocardiographic parameter ( ejection fraction ) of individual Cre control ( CLIP - 170 S311A + / + ; α - MHC - MerCreMer + / − ) , Control mice ( CLIP - 170 S311A flox / + ; α - MHC - MerCreMer − / − ) , and CLIP - 170 S311A overexpressing mice ( CLIP - 170 S311A flox / + ; α - MHC - MerCreMer + / − ) , before ( Pre ) , 2 weeks ( Tx2w ) , 8 weeks ( Tx8w ) , 26 weeks ( Tx26w ) and over 1 year ( Tx1y ) after tamoxifen induction . Number of mice , ( Cre control , Control , S311A ) , Pre ( 6 , 2 , 3 ) , Tx 2w ( 9 , 5 , 5 ) , Tx 8w ( 9 , 10 , 14 ) , Tx1 2w ( 7 , 10 , 14 ) , Tx 26w ( 3 , 7 , 8 ) , Tx 52w ( 3 , 7 , 9 ) .

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

(A) Beeswarm plots of the echocardiographic parameter (ejection fraction) of individual Cre control (CLIP-170 S311A +/+; α-MHC-MerCreMer+/−), Control mice (CLIP-170 S311A flox/+; α-MHC-MerCreMer−/−), and CLIP-170 S311A overexpressing mice (CLIP-170 S311A flox/+; α-MHC-MerCreMer+/−), before (Pre), 2 weeks (Tx2w), 8 weeks (Tx8w), 26 weeks (Tx26w) and over 1 year (Tx1y) after tamoxifen induction. Number of mice, (Cre control, Control, S311A), Pre (6, 2, 3), Tx 2w (9, 5, 5), Tx 8w (9, 10, 14), Tx1 2w (7, 10, 14), Tx 26w (3, 7, 8), Tx 52w (3, 7, 9).


( B ) Beeswarm plots of another line of CLIP - 170 S311A overexpressing mice ( line 9 ) . Number of mice , ( Control , S311A ) , Pre ( 7 , 3 ) , Tx 2w ( 4 , 7 ) , Tx 8w ( 6 , 4 ) , Tx 12w ( 5 , 4 ) , Tx 52w ( 3 , 3 ) . Data means ± S . D . Statistical significance was determined by two - way ANOVA followed by post hoc Tukey ' s multiple comparison test . # # : p < 0 . 01 vs Duration - matched Cre Control , * : p < 0 . 05 , * * : p < 0 . 01 vs Duration - matched Control ; † : p < 0 . 05 , † † : p < 0 . 01 , S311A TG mice 12 weeks vs 52 weeks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Beeswarm plots of another line of CLIP-170 S311A overexpressing mice (line 9). Number of mice, (Control, S311A), Pre (7, 3), Tx 2w (4, 7), Tx 8w (6, 4), Tx 12w (5, 4), Tx 52w (3, 3). Data means ±S.D. Statistical significance was determined by two-way ANOVA followed by post hoc Tukey's multiple comparison test. ##: p < 0.01 vs Duration-matched Cre Control, *: p < 0.05, **: p < 0.01 vs Duration-matched Control; †: p < 0.05, ††: p < 0.01, S311A TG mice 12 weeks vs 52 weeks.


( C ) Representative long axis four - chamber cardiac magnetic resonance images of CLIP - 170 S311A TG mice and control mice over 1 year after tamoxifen induction . Left column showed systole images and right column represented diastole images . Three mice from each group were analyzed for cardiac MRI . The representative images were shown .

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

(C) Representative long axis four-chamber cardiac magnetic resonance images of CLIP-170 S311A TG mice and control mice over 1 year after tamoxifen induction. Left column showed systole images and right column represented diastole images. Three mice from each group were analyzed for cardiac MRI. The representative images were shown.


( D ) Beeswarm plots showed the ejection fraction from cardiac MRI . LV , left ventricular , RV , right ventricular . Number of mice , ( Control , S311A ) , 1y ( 3 , 3 ) . Data means ± S . D . Two - tailed Student ' s t - test was used to analyze differences between two groups . * * , P < 0 . 01 versus Control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Beeswarm plots showed the ejection fraction from cardiac MRI. LV, left ventricular, RV, right ventricular. Number of mice, (Control, S311A), 1y (3, 3). Data means ±S.D. Two-tailed Student's t-test was used to analyze differences between two groups. **, P<0.01 versus Control.


( E ) Beeswarm plots of the echocardiographic parameter ( ejection fraction ) from CLIP - 170 S311D overexpressing mice and the control . Number of mice , ( Control , S311D ) , Tx 2w ( 3 , 4 ) , Tx 8w ( 5 , 11 ) , Tx 26w ( 4 , 11 ) . Data means ± S . D . There was no significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Beeswarm plots of the echocardiographic parameter (ejection fraction) from CLIP-170 S311D overexpressing mice and the control. Number of mice, (Control, S311D), Tx 2w (3, 4), Tx 8w (5, 11), Tx 26w (4, 11). Data means ±S.D. There was no significant difference.


( A ) Masson ' s trichrome staining of the heart of CLIP - 170 S311A overexpressing mice and the control mice from 3 months and 1 year after tamoxifen treatment .

O O O O O O O O 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) Masson's trichrome staining of the heart of CLIP-170 S311A overexpressing mice and the control mice from 3 months and 1 year after tamoxifen treatment.


( B ) Quantitative analysis of cardiac fibrosis . Quantification was made as percentage of fibrosis area ( in blue ) in the left ventricle . Number of mice , ( Control , S311A ) , 3m ( 7 , 6 ) , 1y ( 5 , 6 ) . * , P < 0 . 05 versus Control .

(B) Quantitative analysis of cardiac fibrosis. Quantification was made as percentage of fibrosis area (in blue) in the left ventricle. Number of mice, (Control, S311A), 3m (7, 6), 1y (5, 6). *, P<0.05 versus Control.


( C ) Representative immunostained images with an α - tubulin antibody of CLIP - 170 S311A overexpressing mice heart and control mice heart over 1 year after tamoxifen treatment . ( D ) Quantitative analysis of intensity of α - tubulin in ( C ) . Number of mice , ( Control : n = 7 , S311A : n = 8 ) . Average of 5 sections per animal were analyzed . * * , P < 0 . 01 versus Control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative immunostained images with an α-tubulin antibody of CLIP-170 S311A overexpressing mice heart and control mice heart over 1 year after tamoxifen treatment. (D) Quantitative analysis of intensity of α-tubulin in (C). Number of mice, (Control: n=7, S311A: n=8). Average of 5 sections per animal were analyzed. **, P<0.01 versus Control.


( E ) Representative images of western blot for an α - tubulin antibody . GAPDH was used as a loading control . ( F ) Quantitative analysis of intensity of α - tubulin in ( E ) . Number of mice , ( Control : n = 4 , S311A : n = 5 ) . * , P < 0 . 05 versus Control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of western blot for an α-tubulin antibody. GAPDH was used as a loading control. (F) Quantitative analysis of intensity of α-tubulin in (E). Number of mice, (Control: n=4, S311A: n=5). *, P<0.05 versus Control.


( G ) Representative immunostained images of CLIP - 170 S311A overexpressing mice heart and the control mice heart over 1 year after tamoxifen treatment . These were stained with WGA ( red ) and a plakoglobin antibody ( green ) .

O O O O O O O O O O O O O O O O O O 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) Representative immunostained images of CLIP-170 S311A overexpressing mice heart and the control mice heart over 1 year after tamoxifen treatment. These were stained with WGA (red) and a plakoglobin antibody (green).


( H ) Quantitative analysis of ( G ) Cell width , length and aspect ratio ( length / width ) were measured . Number of cells analyzed , Control , n = 251 , S311A , n = 310 , * * , P < 0 . 01 versus Control .

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

(H) Quantitative analysis of (G) Cell width, length and aspect ratio (length/width) were measured. Number of cells analyzed, Control, n=251, S311A, n=310, **, P<0.01 versus Control.


Verification of PTPδ - tdTomato reporter mice by Western blot analysis of whole - brain lysates from heterozygote ( HT ) and homozygous ( Ho ) mice ( P21 ) and PTPδ using mCherry antibodies . Note that levels of endogenous PTPδ protein ( ~ 85 kDa ) are decreased , and that PTPδ - tdTomato protein ( ~ 140 kDa ) is detectable only in reporter mice ( HT and Ho ) . Note also that the 85 - kDa band represents the transmembrane domain + cytoplasmic fragment of the full - length PTPδ protein that undergoes proteolytic cleavage at extracellular membrane - proximal sites , which can only be recognized by the PTPδ C - terminal antibody that targets the cytoplasmic region . The PTPδ - tdTomato protein ( ~ 140 kDa ) contains the membrane domain + cytoplasmic fragment of PTPδ ( ~ 85 kDa ) fused to tdTomato ( 55 kDa ) . The N - terminal antibody recognizes equal amounts of the C - terminal cleavage product derived from PTPδ and PTPδ - tdTomato proteins .

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

Verification of PTPδ-tdTomato reporter mice by Western blot analysis of whole-brain lysates from heterozygote (HT) and homozygous (Ho) mice (P21) and PTPδ using mCherry antibodies. Note that levels of endogenous PTPδ protein (~85 kDa) are decreased, and that PTPδ-tdTomato protein (~140 kDa) is detectable only in reporter mice (HT and Ho). Note also that the 85-kDa band represents the transmembrane domain + cytoplasmic fragment of the full-length PTPδ protein that undergoes proteolytic cleavage at extracellular membrane-proximal sites, which can only be recognized by the PTPδ C-terminal antibody that targets the cytoplasmic region. The PTPδ-tdTomato protein (~140 kDa) contains the membrane domain + cytoplasmic fragment of PTPδ (~85 kDa) fused to tdTomato (55 kDa). The N-terminal antibody recognizes equal amounts of the C-terminal cleavage product derived from PTPδ and PTPδ-tdTomato proteins.


Representative coronal , horizontal , and sagittal sections from PTPδ - tdTomato mice . Enlarged windows are demarcated by black dotted lines ( Fig EV1A - C ) . Red tdTomato images were converted to grayscale images for clarity . ACA , anterior cingulate area ; ACB , nucleus accumbens ; BLA , basolateral amygdala ; BMA , basomedial amygdala ; CA1 , cornu ammonis area 1 ; CA2 , cornu ammonis area 2 ; CA3 , cornu ammonis area 3 ; CB , cerebellum ; CC , corpus callosum ; CEA , central amygdala ; CP , caudate putamen ; CST , corticospinal tract ; DG , dentate gyrus ; EP , endopiriform nucleus , HY , hypothalamus ; L1 , cortical layer 1 ; L2 / 3 , cortical layer 2 / 3 ; LA , lateral amygdala ; lENT , lateral entorhinal area ; LH , lateral habenula ; MB , midbrain ; MEA , medial amygdala ; mENT , medial entorhinal area ; MH , medial habenula ; PIR1 , piriform molecular layer ; PIR2 , piriform pyramidal layer ; PIR3 , piriform polymorph layer ; PL , prelimbic ; PONS , pons ; PRE , presubiculum ; RSP , retrosplenial area ; SS , somatosensory area ; SUB , subiculum ; TEa , temporal association area ; TH , thalamus ; VIS , visual area ; Scale bar , 500 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 coronal, horizontal, and sagittal sections from PTPδ-tdTomato mice. Enlarged windows are demarcated by black dotted lines (Fig EV1A-C). Red tdTomato images were converted to grayscale images for clarity. ACA, anterior cingulate area; ACB, nucleus accumbens; BLA, basolateral amygdala; BMA, basomedial amygdala; CA1, cornu ammonis area 1; CA2, cornu ammonis area 2; CA3, cornu ammonis area 3; CB, cerebellum; CC, corpus callosum; CEA, central amygdala; CP, caudate putamen; CST, corticospinal tract; DG, dentate gyrus; EP, endopiriform nucleus, HY, hypothalamus; L1, cortical layer 1; L2/3, cortical layer 2/3; LA, lateral amygdala; lENT, lateral entorhinal area; LH, lateral habenula; MB, midbrain; MEA, medial amygdala; mENT, medial entorhinal area; MH, medial habenula; PIR1, piriform molecular layer; PIR2, piriform pyramidal layer; PIR3, piriform polymorph layer; PL, prelimbic; PONS, pons; PRE, presubiculum; RSP, retrosplenial area; SS, somatosensory area; SUB, subiculum; TEa, temporal association area; TH, thalamus; VIS, visual area; Scale bar, 500 μm.


PTPδ - tdTomato protein is detected in tau - positive axonal compartments , but not in MAP2 - positive dendritic compartments in cultured neurons ( entorhinal cortex + hippocampus ; 1 : 2 mixture ; days in vitro or DIV 24 ) derived from Ptprd - / - mouse embryos . Note that the tdTomato signals envelop , but not overlap with , MAP2 - delineated dendrites , although this does not necessarily suggest that tdTomato signals not in dendrites . Scale bars , 10 μm in main images and 2 μm in enlarged images .

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

PTPδ-tdTomato protein is detected in tau-positive axonal compartments, but not in MAP2-positive dendritic compartments in cultured neurons (entorhinal cortex + hippocampus; 1:2 mixture; days in vitro or DIV 24) derived from Ptprd-/- mouse embryos. Note that the tdTomato signals envelop, but not overlap with, MAP2-delineated dendrites, although this does not necessarily suggest that tdTomato signals not in dendrites. Scale bars, 10 μm in main images and 2 μm in enlarged images.


Presynaptic PTPδ - tdTomato clusters colocalize more strongly with PSD - 95 clusters ( excitatory postsynaptic marker ) relative to gephyrin ( inhibitory postsynaptic marker ) clusters in cultured neurons ( entorhinal cortex + hippocampus ; DIV24 ) derived from Ptprd - / - mouse embryos . See Fig EV1F for the quantification of the results . Scale bars , 10 μm in main images and 2 μm in enlarged images .

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

Presynaptic PTPδ-tdTomato clusters colocalize more strongly with PSD-95 clusters (excitatory postsynaptic marker) relative to gephyrin (inhibitory postsynaptic marker) clusters in cultured neurons (entorhinal cortex + hippocampus; DIV24) derived from Ptprd-/- mouse embryos. See Fig EV1F for the quantification of the results. Scale bars, 10 μm in main images and 2 μm in enlarged images.


Ultrastructural localization of PTPδ - tdTomato protein ( red arrows ; DAB staining ) at axon terminals juxtaposed to electron - dense postsynaptic densities ( purple arrows ; PSDs ) and also at vGlut2 - positive excitatory synaptic axon terminals ( green arrows ; immunogold staining ) but not at GAD67 / 65 - positive inhibitory synaptic axon terminals ( blue arrows ; immunogold staining ) in the stratum lacunosum - moleculare ( SLM ) region of the CA1 hippocampal region ( P21 ) . Note that ~ 85 % of PTPδ - tdTomato signals are present in excitatory presynaptic terminals apposed to PSDs , ~ 10 % at presynaptic terminals not apposed to PSDs , and ~ 5 % in neuropils other than presynaptic terminals . Axon terminals are indicated by pink shades . Scale bar , 500 nm . ( n = 4 areas from 2 mice [ WT and KO ] ) .

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

Ultrastructural localization of PTPδ-tdTomato protein (red arrows; DAB staining) at axon terminals juxtaposed to electron-dense postsynaptic densities (purple arrows; PSDs) and also at vGlut2-positive excitatory synaptic axon terminals (green arrows; immunogold staining) but not at GAD67/65-positive inhibitory synaptic axon terminals (blue arrows; immunogold staining) in the stratum lacunosum-moleculare (SLM) region of the CA1 hippocampal region (P21). Note that ~85% of PTPδ-tdTomato signals are present in excitatory presynaptic terminals apposed to PSDs, ~10% at presynaptic terminals not apposed to PSDs, and ~5% in neuropils other than presynaptic terminals. Axon terminals are indicated by pink shades. Scale bar, 500 nm. (n = 4 areas from 2 mice [WT and KO]).


Strong PTPδ - tdTomato ( red ) signals in the SLM layer of the CA1 region and the MO layer of the DG in the hippocampus ( 2 months ) , revealed by quantitative analysis of PTPδ - tdTomato fluorescence across hippocampal lamina . DAPI was used for nuclear staining . CC , corpus callosum ; SO , stratum oriens ; SP , stratum pyramidale ; SR , stratum radiatum ; SLM , stratum lacunosum moleculare ; MO , molecular layer ; SG , granule cell layer ; PO , polymorph layer . Scale bar 500 μm .

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

Strong PTPδ-tdTomato (red) signals in the SLM layer of the CA1 region and the MO layer of the DG in the hippocampus (2 months), revealed by quantitative analysis of PTPδ-tdTomato fluorescence across hippocampal lamina. DAPI was used for nuclear staining. CC, corpus callosum; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunosum moleculare; MO, molecular layer; SG, granule cell layer; PO, polymorph layer. Scale bar 500 μm.


Biochemical enrichment of PTPδ protein in the hippocampal SLM layer relative to the SR layer . Micro - dissected SLM and SR lysates ( 2 months ) were immunoblotted for PTPδ ( C - terminal antibodies ) and α - tubulin ( control ) . The asterisk indicates a non - specific band . Analysis of immunoblots reveal significant enrichment of PTPδ in the SLM compared to the SR . ( n = 4 mice for SR and SLM , mean ± SEM , * * * p < 0 . 001 , Student ' s t - test ; See Dataset EV2 for statistical details for these and all following results ) .

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

Biochemical enrichment of PTPδ protein in the hippocampal SLM layer relative to the SR layer. Micro-dissected SLM and SR lysates (2 months) were immunoblotted for PTPδ (C-terminal antibodies) and α-tubulin (control). The asterisk indicates a non-specific band. Analysis of immunoblots reveal significant enrichment of PTPδ in the SLM compared to the SR. (n = 4 mice for SR and SLM, mean ± SEM, ***p < 0.001, Student's t-test; See Dataset EV2 for statistical details for these and all following results).


Gene dose - dependent reductions in PTPδ protein levels in whole brains of Ptprd + / - ( HT ) and Ptprd - / - ( KO ) mice relative to Ptprd + / + ( WT ) mice ( P21 ) , shown by immunoblot analyses . Note that the C - terminal antibody also detects a non - specific band of similar size indicated by an asterisk , possibly either PTPσ or LAR .

Gene dose-dependent reductions in PTPδ protein levels in whole brains of Ptprd+/- (HT) and Ptprd-/- (KO) mice relative to Ptprd+/+ (WT) mice (P21), shown by immunoblot analyses. Note that the C-terminal antibody also detects a non-specific band of similar size indicated by an asterisk, possibly either PTPσ or LAR.


Suppressed basal excitatory synaptic transmission in the Ptprd - / - hippocampal SLM layer , but not SO or SR layer ( 2 months ; male ) , as shown by the I / O ratio ( n = 14 slices from 5 mice [ WT - SO ] , 14 , 5 [ KO - SO ] , 11 , 4 [ WT - SR ] , 12 , 5 [ KO - SR ] , 14 , 6 [ WT - SLM ] , 12 , 6 [ KO - SLM ] , mean ± SEM , * p < 0 . 05 ; * * p < 0 . 01 , two - way repeated - measures / RM ANOVA , Holm - Sidak post - hoc multiple comparisons test ) . ( F , H , and J ) Normal presynaptic release in the Ptprd - / - hippocampal SLM , SO , and SR layers ( P22 - 27 ; male ) , as shown by the PPF ratio ( n = 13 slices from 5 mice [ WT - SO ] , 13 , 5 [ KO - SO ] , 10 , 4 [ WT - SR ] , 12 , 5 [ KO - SR ] , 15 , 6 [ WT - SLM ] , 14 , 6 [ KO - SLM ] , mean ± SEM , ns , not significant , two - way RM ANOVA ) .

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

Suppressed basal excitatory synaptic transmission in the Ptprd-/- hippocampal SLM layer, but not SO or SR layer (2 months; male), as shown by the I/O ratio (n = 14 slices from 5 mice [WT-SO], 14, 5 [KO-SO], 11, 4 [WT-SR], 12, 5 [KO-SR], 14, 6 [WT-SLM], 12, 6 [KO-SLM], mean ± SEM, *p < 0.05; **p < 0.01, two-way repeated-measures/RM ANOVA, Holm-Sidak post-hoc multiple comparisons test). (F, H, and J) Normal presynaptic release in the Ptprd-/- hippocampal SLM, SO, and SR layers (P22-27; male), as shown by the PPF ratio (n = 13 slices from 5 mice [WT-SO], 13, 5 [KO-SO], 10,4 [WT-SR], 12, 5 [KO-SR], 15, 6 [WT-SLM], 14, 6 [KO-SLM], mean ± SEM, ns, not significant, two-way RM ANOVA).


Decreased PSD density , but not PSD length , thickness , or perforation ( % ) , in the Ptprd - / - SLM ( P21 ; male ) , revealed by EM . Arrows in panel K indicate PSDs .

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

Decreased PSD density, but not PSD length, thickness, or perforation (%), in the Ptprd-/- SLM (P21; male), revealed by EM. Arrows in panel K indicate PSDs.


Decreased PSD density , but not PSD length , thickness , or perforation ( % ) , in the Ptprd - / - SLM ( P21 ; male ) , revealed by EM . Arrows in panel K indicate PSDs . ( n = 18 images from 3 mice [ WT and KO ] , * * * p < 0 . 001 , ns , not significant , Student ' s t - test for L , O - R , Mann - Whitney U test for M , N ) . Scale bar , 500 nm .

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

Decreased PSD density, but not PSD length, thickness, or perforation (%), in the Ptprd-/- SLM (P21; male), revealed by EM. Arrows in panel K indicate PSDs. (n = 18 images from 3 mice [WT and KO], ***p < 0.001, ns, not significant, Student's t-test for L, O-R, Mann-Whitney U test for M, N). Scale bar, 500 nm.


Decreased density of presynaptic nerve terminals but normal area of nerve terminals and normal density of presynaptic vesicles in the Ptprd - / - SLM ( P21 ; male ) , revealed by EM . ( n = 3 mice [ WT and KO ] , mean ± SEM , * * * p < 0 . 001 , ns , not significant , Student ' s t - test ) .

Decreased density of presynaptic nerve terminals but normal area of nerve terminals and normal density of presynaptic vesicles in the Ptprd-/- SLM (P21; male), revealed by EM. (n = 3 mice [WT and KO], mean ± SEM, ***p < 0.001, ns, not significant, Student's t-test).


A volcano plot of proteins that showed significant up - or downregulation of pTyr levels ( absolute fold change > 1 . 5 , p < 0 . 05 ) in the whole brain of Ptprd - / - mice ( P21 ) . Note that IL1RAPL1 is the protein with the strongest decrease in pTyr levels . ( n = 3 for WT and 3 for KO ) . See Dataset EV1 , ' Phosphoscan Results ' tab for a full list of significantly changed proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 volcano plot of proteins that showed significant up- or downregulation of pTyr levels (absolute fold change > 1.5, p < 0.05) in the whole brain of Ptprd-/- mice (P21). Note that IL1RAPL1 is the protein with the strongest decrease in pTyr levels. (n = 3 for WT and 3 for KO). See Dataset EV1, 'Phosphoscan Results' tab for a full list of significantly changed proteins.


A volcano plot of presynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels ( p < 0 . 05 ) .

O O O O O 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 volcano plot of presynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels (p < 0.05).


A volcano plot of postsynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels ( p < 0 . 05 ) .

A volcano plot of postsynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels (p < 0.05).


Decreased pTyr levels , but normal total levels of IL1RAPL1 protein , in the Ptprd - / - brain ( P21 - 27 ) , as revealed by immunoprecipitation of IL1RAPL1 from whole - brain lysates followed by immunoblotting for total IL1RAPL1 protein and pTyr levels ( 4G10 antibody ) .

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

Decreased pTyr levels, but normal total levels of IL1RAPL1 protein, in the Ptprd-/- brain (P21-27), as revealed by immunoprecipitation of IL1RAPL1 from whole-brain lysates followed by immunoblotting for total IL1RAPL1 protein and pTyr levels (4G10 antibody).


Decreased levels of synaptic IL1RAPL1 protein in the Ptprd - / - brain ( P21 - 27 ) , as revealed by immunoblotting of crude synaptosomal ( P2 ) , synaptic plasma membrane ( SPM ) , and PSD ( PSD II ) fractions . Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd - / - brain samples . ( n = 3 mice [ WT and KO ] for each fraction [ P2 , SPM , and PSD ] , mean ± SEM , * * p < 0 . 01 , 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

Decreased levels of synaptic IL1RAPL1 protein in the Ptprd-/- brain (P21-27), as revealed by immunoblotting of crude synaptosomal (P2), synaptic plasma membrane (SPM), and PSD (PSD II) fractions. Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd-/- brain samples. (n = 3 mice [WT and KO] for each fraction [P2, SPM, and PSD], mean ± SEM, **p<0.01, Student's t-test).


Normal levels of the PTPδ protein in whole - brain lysates of Ptprd - meA - / - mice ( P21 - 27 ) , which contrasts with the Ptprd - / - brain , where PTPδ protein is undetectable .

Normal levels of the PTPδ protein in whole-brain lysates of Ptprd-meA-/- mice (P21-27), which contrasts with the Ptprd-/- brain, where PTPδ protein is undetectable.


Strongly decreased levels of tyrosine - phosphorylated PTPδ protein in whole - brain lysates of Ptprd - meA - / - mice ( P21 - 27 ) , as shown by the immunoprecipitation of IL1RAPL1 protein from whole - brain lysates followed by immunoblotting for total and tyrosine - phosphorylated PTPδ protein .

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 B-MEASURED_VAR O O O O O

Strongly decreased levels of tyrosine-phosphorylated PTPδ protein in whole-brain lysates of Ptprd-meA-/- mice (P21-27), as shown by the immunoprecipitation of IL1RAPL1 protein from whole-brain lysates followed by immunoblotting for total and tyrosine-phosphorylated PTPδ protein.


Decreased levels of synaptic IL1RAPL1 protein in the Ptprd - meA - / - brain ( P21 - 27 ) , as revealed by immunoblotting P2 , SPM , and PSD ( PSD II ) fractions . Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd - meA - / - brain samples . ( n = 3 mice [ Ptprd - meA + / + and Ptprd - meA - / - ] and [ P2 , SPM , and PSD ] , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 001 , Student ' s t - test ) . Note that , while unchanged in the P2 and SPM fractions , PTPδ is significantly decreased in the PSD of Ptprd - meA - / - samples .

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

Decreased levels of synaptic IL1RAPL1 protein in the Ptprd-meA-/- brain (P21-27), as revealed by immunoblotting P2, SPM, and PSD (PSD II) fractions. Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd-meA-/- brain samples. (n = 3 mice [Ptprd-meA+/+ and Ptprd-meA-/-] and [P2, SPM, and PSD], mean ± SEM, *p<0.05, ***p < 0.001, Student's t-test). Note that, while unchanged in the P2 and SPM fractions, PTPδ is significantly decreased in the PSD of Ptprd-meA-/- samples.


Decreased basal excitatory synaptic transmission in the hippocampal SLM region of Ptprd - meA - / - mice , as shown by the I / O ratio at the TA - SLM pathway ( P20 - 24 ) . ( n = 15 slice from 5 mice [ Ptprd - meA + / + ] , 12 , 4 [ Ptprd - meA - / - ] , mean ± SEM , * p < 0 . 05 ; * * * p < 0 . 001 , two - way RM ANOVA with Holm - Sidak 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

Decreased basal excitatory synaptic transmission in the hippocampal SLM region of Ptprd-meA-/- mice, as shown by the I/O ratio at the TA-SLM pathway (P20-24). (n = 15 slice from 5 mice [Ptprd-meA+/+], 12, 4 [Ptprd-meA-/-], mean ± SEM, *p < 0.05; ***p<0.001, two-way RM ANOVA with Holm-Sidak test).


Normal presynaptic release in the hippocampal SLM region of Ptprd - meA - / - mice , as shown by the PPF ratio at the TA - SLM pathway ( P20 - 24 ) . ( n = 15 slice from 5 mice [ Ptprd - meA + / + ] and 13 , 4 [ Ptprd - meA - / - ] , mean ± SEM , ns , not significant , two - way RM ANOVA ) .

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

Normal presynaptic release in the hippocampal SLM region of Ptprd-meA-/- mice, as shown by the PPF ratio at the TA-SLM pathway (P20-24). (n = 15 slice from 5 mice [Ptprd-meA+/+] and 13, 4 [Ptprd-meA-/-], mean ± SEM, ns, not significant, two-way RM ANOVA).


Hyperactivity of Ptprd - / - mice ( 2 . 5 - 4 months ) in Laboras cages observed during light - off and light - on periods were strongly mimicked by Emx1 - cKO mice , but more weakly by Viaat - cKO mice and not at all by Camk2a - cKO mice . Note also that immobility , a measure that better reflects sleep behavior , showed similar changes among different mouse lines . ( n = 14 mice [ WT ] , 14 [ global KO ] , 16 [ Emx1 - cWT ] , 16 [ Emx1 - cKO ] , 11 [ Viaat - cWT ] , 13 [ Viaat - cKO ] , 18 [ Camk2a - cWT ] and 13 [ Camk2a - cKO ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; ns , not significant , two - way RM ANOVA with Holm - Sidak 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hyperactivity of Ptprd-/- mice (2.5-4 months) in Laboras cages observed during light-off and light-on periods were strongly mimicked by Emx1-cKO mice, but more weakly by Viaat-cKO mice and not at all by Camk2a-cKO mice. Note also that immobility, a measure that better reflects sleep behavior, showed similar changes among different mouse lines. (n = 14 mice [WT], 14 [global KO], 16 [Emx1-cWT], 16 [Emx1-cKO], 11 [Viaat-cWT], 13 [Viaat-cKO], 18 [Camk2a-cWT] and 13 [Camk2a-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Hyperactive behavior and decreased immobility of Ptprd - meA - / - mice ( 2 . 5 - 4 months ) in Laboras cages observed during light - off periods and light - on periods , similar to Ptprd - / - mice . ( n = 7 mice for Ptprd - meA + / + and 7 mice for Ptprd - meA - / - , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , two - way RM ANOVA with Holm - Sidak 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

Hyperactive behavior and decreased immobility of Ptprd-meA-/- mice (2.5-4 months) in Laboras cages observed during light-off periods and light-on periods, similar to Ptprd-/- mice. (n = 7 mice for Ptprd-meA+/+ and 7 mice for Ptprd-meA-/-, mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Increased WAKE duration and decreased NREM duration during the first three hours of the light - on period in Emx1 - Cre ; Ptprdfl / fl mice ( 2 . 5 - 4 months ) . ( n = 9 mice [ Emx1 - cWT ] and 9 mice [ Emx1 - cKO ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , ns , not significant , two - way RM ANOVA with Holm - Sidak 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

Increased WAKE duration and decreased NREM duration during the first three hours of the light-on period in Emx1-Cre;Ptprdfl/fl mice (2.5-4 months). (n = 9 mice [Emx1-cWT] and 9 mice [Emx1-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Decreased delta power ( normalized to total power ) in NREM , but not WAKE , oscillations in Emx1 - Cre ; Ptprdfl / fl mice ( 2 . 5 - 4 months ) . Note also the normal theta power ( normalized to total power ) in REM oscillations in Emx1 - Cre ; Ptprdfl / fl mice . ( n = 9 [ Emx1 - cWT ] and 9 [ Emx1 - cKO ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , ns , not significant , two - way RM ANOVA with Holm - Sidak 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

Decreased delta power (normalized to total power) in NREM, but not WAKE, oscillations in Emx1-Cre;Ptprdfl/fl mice (2.5-4 months). Note also the normal theta power (normalized to total power) in REM oscillations in Emx1-Cre;Ptprdfl/fl mice. (n = 9 [Emx1-cWT] and 9 [Emx1-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


( D ) Increased WAKE duration and decreased NREM duration during the first three hours of the light - on period and decreased REM duration in the first half of the light - on period in Ptprd - meA - / - mice ( 2 . 5 - 4 months ) . Note also that Ptprd - meA - / - mice also display increased WAKE duration and decreased NREM duration during the light - off period . ( n = 8 [ Ptprd - meA + / + ] and 8 [ Ptprd - meA - / - ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , two - way RM ANOVA with Holm - Sidak 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 O O O O O O O O O O O O O O O

(D) Increased WAKE duration and decreased NREM duration during the first three hours of the light-on period and decreased REM duration in the first half of the light-on period in Ptprd-meA-/- mice (2.5-4 months). Note also that Ptprd-meA-/- mice also display increased WAKE duration and decreased NREM duration during the light-off period. (n = 8 [Ptprd-meA+/+] and 8 [Ptprd-meA-/-], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Decreased delta power ( normalized to total power ) in NREM ( both light - off and light - on periods ) and WAKE ( light - on period ) oscillations , and decreased theta power in REM ( mainly light - on period ) oscillations in Ptprd - meA - / - mice ( 2 . 5 - 4 months ) . ( n = 8 [ Ptprd - meA + / + ] and 8 [ Ptprd - meA - / - ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , two - way RM ANOVA with Holm - Sidak 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

Decreased delta power (normalized to total power) in NREM (both light-off and light-on periods) and WAKE (light-on period) oscillations, and decreased theta power in REM (mainly light-on period) oscillations in Ptprd-meA-/- mice (2.5-4 months). (n = 8 [Ptprd-meA+/+] and 8 [Ptprd-meA-/-], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


The percentage of myofibroblast - like cells in total endometrial cells between controls and patients with IUA ( n = 3 samples for each group ) . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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

The percentage of myofibroblast-like cells in total endometrial cells between controls and patients with IUA (n=3 samples for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.


Masson trichrome staining and immunostaining of α - SMA and Collagen 1 in endometrium of controls and patients with IUA ( n = 5 samples for each group ) . Scale bars : 100μm . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

Masson trichrome staining and immunostaining of α-SMA and Collagen 1 in endometrium of controls and patients with IUA (n=5 samples for each group). Scale bars: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.


Expression of marker genes for each cell subtype of myofibroblast - like cells ( n = 6 samples ) .

Expression of marker genes for each cell subtype of myofibroblast-like cells (n=6 samples).


Flow cytometric analysis of the proportion of CD14 + cells , CD301 + macrophages , CD80 + macrophages and CD163 + macrophages in the endometrium of normal controls and patients with IUA . Macro , macrophage ( n = 20 samples for each group ) . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant .

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

Flow cytometric analysis of the proportion of CD14+ cells, CD301+ macrophages, CD80+ macrophages and CD163+ macrophages in the endometrium of normal controls and patients with IUA. Macro, macrophage (n=20 samples for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.


Representative immunostaining images of CD301 and CD68 from samples of normal controls and patients with IUA ( n = 5 samples for each group ) . Representative immunostaining images of CD301 and α - SMA from samples of normal controls and patients with IUA ( n = 5 samples for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant .

Representative immunostaining images of CD301 and CD68 from samples of normal controls and patients with IUA (n=5 samples for each group). Representative immunostaining images of CD301 and α-SMA from samples of normal controls and patients with IUA (n=5 samples for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.


The protein levels of CD14 and CD301 in CD301 - macrophages and CD301 + macrophages examined by western blot ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant .

The protein levels of CD14 and CD301 in CD301- macrophages and CD301+ macrophages examined by western blot (n=3 technical replicates for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.


The protein levels of Fibronectin , Collagen 1 , α - SMA and CTGF expression in primary human endometrial stromal cells ( hESCs ) treated by the supernatants of CD301 - and CD301 + macrophages for 48h , respectively examined by western blot ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant .

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

The protein levels of Fibronectin, Collagen 1, α-SMA and CTGF expression in primary human endometrial stromal cells (hESCs) treated by the supernatants of CD301- and CD301+ macrophages for 48h, respectively examined by western blot (n=3 technical replicates for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.


The protein levels of GAS6 and CCL8 expression in sorted human endometrial CD301 - and CD301 + macrophages ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant .

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

The protein levels of GAS6 and CCL8 expression in sorted human endometrial CD301- and CD301+ macrophages (n=3 technical replicates for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.


Localization of GAS6 and CD301 in endometrium of normal controls and patients with IUA examined by immunofluorescence ( n = 5 samples for each group ) . Localization of AXL and α - SMA in endometrium of normal controls and patients with IUA examined by immunofluorescence ( n = 5 samples for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant .

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

Localization of GAS6 and CD301 in endometrium of normal controls and patients with IUA examined by immunofluorescence (n=5 samples for each group). Localization of AXL and α-SMA in endometrium of normal controls and patients with IUA examined by immunofluorescence (n=5 samples for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.


Western blot analysis of phospho - AXL ( p - AXL ) and AXL in hESCs treated with GAS6 at dose of 10 , 20 , 50 , 100 and 200 ng / mL for 72h ( n = 3 technical replicates for each group ) . The protein levels of Collagen 1 and α - SMA expression in hESCs after treatment with GAS6 at dose of 10 , 20 , 50 , 100 and 200 ng / mL for 72h examined by western blot ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant .

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

Western blot analysis of phospho-AXL (p-AXL) and AXL in hESCs treated with GAS6 at dose of 10, 20, 50, 100 and 200 ng/mL for 72h (n=3 technical replicates for each group). The protein levels of Collagen 1 and α-SMA expression in hESCs after treatment with GAS6 at dose of 10, 20, 50, 100 and 200 ng/mL for 72h examined by western blot (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.


Western blot analysis of α - SMA and Collagen 1 in hESCs treated with Bemcentinib ( 1μM ) for 1 hour , followed by GAS6 ( 50ng / mL ) for 72h ( n = 3 technical replicates for each group ) . Western blot analysis of α - SMA and Collagen 1 in hESCs treated with the supernatants of CD301 - and CD301 + macrophages and GAS6 neutralizing antibody ( 2 . 5μg / mL ) for 72h ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . NS , not significant .

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

Western blot analysis of α-SMA and Collagen 1 in hESCs treated with Bemcentinib (1μM) for 1 hour, followed by GAS6 (50ng/mL) for 72h (n=3 technical replicates for each group). Western blot analysis of α-SMA and Collagen 1 in hESCs treated with the supernatants of CD301- and CD301+ macrophages and GAS6 neutralizing antibody (2.5μg/mL) for 72h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.


Western blot analysis of phospho - p65 ( p - p65 ) and p65 expression in hESCs treated with GAS6 ( 50ng / mL ) at time of 3 , 6 , 12 , 24 and 48h ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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

Western blot analysis of phospho-p65 (p-p65) and p65 expression in hESCs treated with GAS6 (50ng/mL) at time of 3, 6, 12, 24 and 48h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.


Western blot analysis of p - p65 , p65 , Collagen 1 and α - SMA expression in hESCs treated with or without GAS6 neutralizing antibody ( 2 . 5μg / mL ) or Bemcentinib ( 1μM ) for 1 hour , followed by GAS6 protein ( 50ng / mL ) for 72h ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O O O O O O O O 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

Western blot analysis of p-p65, p65, Collagen 1 and α-SMA expression in hESCs treated with or without GAS6 neutralizing antibody (2.5μg/mL) or Bemcentinib (1μM) for 1 hour, followed by GAS6 protein (50ng/mL) for 72h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.


Representative immunostaining images of p - p65 in endometrium of normal controls and patients with IUA ( n = 5 samples for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . ( D ) Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

Representative immunostaining images of p-p65 in endometrium of normal controls and patients with IUA (n=5 samples for each group). Scale bar: 100μm. Data are presented as mean ± SEM. (D) Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.


qRT - PCR analysis of ACTA2 and COL1A1 expression in hESCs treated with GAS6 ( 50ng / mL ) or JSH23 ( 10μM ) for 72h ( n = 3 biological replicates for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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

qRT-PCR analysis of ACTA2 and COL1A1 expression in hESCs treated with GAS6 (50ng/mL) or JSH23 (10μM) for 72h (n=3 biological replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.


Western blot analysis of Collagen 1 and α - SMA expression in hESCs treated with GAS6 ( 50ng / mL ) or JSH23 ( 10μM ) for 72h ( n = 3 technical replicates for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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

Western blot analysis of Collagen 1 and α-SMA expression in hESCs treated with GAS6 (50ng/mL) or JSH23 (10μM) for 72h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.


Representative images of Masson trichrome staining and immunostaining for Collagen 1 between endometrium of Sham and IUA mice ( n = 5 mice for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of Masson trichrome staining and immunostaining for Collagen 1 between endometrium of Sham and IUA mice (n=5 mice for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.


Flow cytometric analysis of the proportions of CD11b + F4 / 80 + cells ( pan - macrophages ) and CD301b + macrophages in the uterus of Sham and IUA mice ( n = 5 mice for each group ) . Macro , macrophage . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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

Flow cytometric analysis of the proportions of CD11b+F4/80+ cells (pan-macrophages) and CD301b+ macrophages in the uterus of Sham and IUA mice (n=5 mice for each group). Macro, macrophage. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.


Representative images of immunostaining for GAS6 ( red ) and CD301b ( green ) in the endometrium of Sham and IUA mice ( n = 5 mice for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . Two - tailed Student ' s t - test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of immunostaining for GAS6 (red) and CD301b (green) in the endometrium of Sham and IUA mice (n=5 mice for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.


Representative images of immunostaining for AXL in the endometrium of Sham and IUA mice ( n = 5 mice for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . ( F ) Mann - Whitney U test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of immunostaining for AXL in the endometrium of Sham and IUA mice (n=5 mice for each group). Scale bar: 100μm. Data are presented as mean ± SEM. (F) Mann-Whitney U test. *, P<0.05; **, P<0.01; ***, P<0.001.


qRT - PCR analysis of Acta2 and Col1a1 expression in the uterus of Sham , IUA and IUA + DT mice ( n = 5 mice for each group ) . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

qRT-PCR analysis of Acta2 and Col1a1 expression in the uterus of Sham, IUA and IUA+DT mice (n=5 mice for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.


Representative images of immunostaining for AXL , Collagen 1 and p - p65 and Masson trichrome staining in the endometria of Sham , IUA and IUA + DT mice ( n = 5 mice for each group ) . Scale bar : 100μm . Data are presented as mean ± SEM . One - way ANOVA with Tukey ' s post hoc analysis . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of immunostaining for AXL, Collagen 1 and p-p65 and Masson trichrome staining in the endometria of Sham, IUA and IUA+DT mice (n=5 mice for each group). Scale bar: 100μm. Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.


qRT - PCR analysis of Acta2 and Col1a1 expression in the uterus of IUA + PBS and IUA + BEM mice ( n = 5 mice for each group ) . Data are presented as mean ± SEM . ( B , Two - tailed Student ' s t - test .

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

qRT-PCR analysis of Acta2 and Col1a1 expression in the uterus of IUA+PBS and IUA+BEM mice (n=5 mice for each group). Data are presented as mean ± SEM. (B, Two-tailed Student's t-test.


Representative images of immunostaining for AXL , Collagen 1 , p - p65 and Masson trichrome staining in the endometrium of IUA + PBS group and IUA + BEM group ( n = 5 mice for each group ) . Scale bar , 100μm . Data are presented as mean ± SEM . C for AXL and Collagen 1 Two - tailed Student ' s t - test . ( C for p - p65 ) Mann - Whitney U test . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O O 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

Representative images of immunostaining for AXL, Collagen 1, p-p65 and Masson trichrome staining in the endometrium of IUA+PBS group and IUA+BEM group (n=5 mice for each group). Scale bar, 100μm. Data are presented as mean ± SEM. C for AXL and Collagen 1 Two-tailed Student's t-test. (C for p-p65) Mann-Whitney U test. *, P<0.05; **, P<0.01; ***, P<0.001.


Loss of LRRC8D causes resistance to carboplatin and cisplatin , but not to oxaliplatin . Survival of parental , vector ‐ transduced , or LRRC8D ‐ deficient GT1 and GT2 KBM7 cells exposed for 96 h to increasing concentrations of cisplatin , carboplatin , and oxaliplatin . The corresponding IC50 values and 95 % confidence interval ( CI ) are given in Appendix Table S2 . Data are presented as mean ± SEM .

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

Loss of LRRC8D causes resistance to carboplatin and cisplatin, but not to oxaliplatin. Survival of parental, vector‐transduced, or LRRC8D‐deficient GT1 and GT2 KBM7 cells exposed for 96 h to increasing concentrations of cisplatin, carboplatin, and oxaliplatin. The corresponding IC50 values and 95% confidence interval (CI) are given in Appendix Table S2. Data are presented as mean ± SEM.


A , BVRAC currents ( ICl , vol ) of the HAP1 ( A ) and KBM7 ( B ) haploid cell lines . Left panels , example current traces of ICl , vol fully activated by hypotonic cell swelling measured with the voltage ‐ clamp protocol shown in ( A ) . Dashed lines indicate zero current . Right panels , averaged current / voltage relationships of maximally activated ICl , vol . Consistent with VRAC currents , they needed hypotonic swelling for activation , displayed an I − > Cl − permeability sequence , and were blocked by DCPIB ( Appendix Fig S2A - H ) . The difference in current inactivation between HAP1 and KBM7 cells can be explained by the fact that KBM7 cells hardly express LRRC8E ( Fig EV1 ) which accelerates VRAC inactivation ( Voss et al , 2014 ) . At potentials > + 100 mV , also KBM7 currents inactivated ( Appendix Fig S2I ) . Data are presented as mean ± SEM ; n = 5 - 10 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, BVRAC currents (ICl,vol) of the HAP1 (A) and KBM7 (B) haploid cell lines. Left panels, example current traces of ICl,vol fully activated by hypotonic cell swelling measured with the voltage‐clamp protocol shown in (A). Dashed lines indicate zero current. Right panels, averaged current/voltage relationships of maximally activated ICl,vol. Consistent with VRAC currents, they needed hypotonic swelling for activation, displayed an I− > Cl− permeability sequence, and were blocked by DCPIB (Appendix Fig S2A-H). The difference in current inactivation between HAP1 and KBM7 cells can be explained by the fact that KBM7 cells hardly express LRRC8E (Fig EV1) which accelerates VRAC inactivation (Voss et al, 2014). At potentials > +100 mV, also KBM7 currents inactivated (Appendix Fig S2I). Data are presented as mean ± SEM; n = 5-10.C


CDependence of regulatory volume decrease ( RVD ) of HEK cells on LRRC8 genes . Cells were exposed to hypotonic medium starting at t = 0 , and intracellular calcein fluorescence was followed over ˜ 1 h as semiquantitative measure of cell volume . Data are presented as mean values ± SEM from sixteen wells .

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

CDependence of regulatory volume decrease (RVD) of HEK cells on LRRC8 genes. Cells were exposed to hypotonic medium starting at t = 0, and intracellular calcein fluorescence was followed over ˜1 h as semiquantitative measure of cell volume. Data are presented as mean values ± SEM from sixteen wells.


LRRC8 subunit ‐ and osmolarity ‐ dependent caspase induction in HCT116 cellsA , BCisplatin ‐ induced caspase activity in the continuous presence of 200 μM cisplatin under isotonic conditions ( A ) , or after 1 h exposure to 200 μM cisplatin under iso ‐ and hypotonic conditions ( B ) , was followed over time in WT , HAP1 − / − , LRRC8D − / − , and LRRC8 − / − HCT116 cells . Results from HAP1 − / − and LRRC8D − / − were obtained with two different clonal cell lines each and averaged . CCaspase activation after 1 ‐ h exposure to 4 μM staurosporine under iso ‐ or hypotonic conditions of WT , HAP1 − / − , LRRC8D − / − , and LRRC8 − / − HCT116 cells . Data information : Data are presented as mean ± SEM , n = 3 - 6 . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 . Similar results were obtained in three independent experiments . Fold change in ( A ) refers to t = 0 . Control experiments indicated that hypotonicity per se had no effect ( Appendix Fig S4 ) .

O 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 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 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

LRRC8 subunit‐ and osmolarity‐dependent caspase induction in HCT116 cellsA, BCisplatin‐induced caspase activity in the continuous presence of 200 μM cisplatin under isotonic conditions (A), or after 1 h exposure to 200 μM cisplatin under iso‐ and hypotonic conditions (B), was followed over time in WT, HAP1−/−, LRRC8D−/−, and LRRC8 −/−HCT116 cells. Results from HAP1−/− and LRRC8D−/− were obtained with two different clonal cell lines each and averaged.CCaspase activation after 1‐h exposure to 4 μM staurosporine under iso‐ or hypotonic conditions of WT, HAP1−/−, LRRC8D−/−, and LRRC8 −/−HCT116 cells.Data information: Data are presented as mean ± SEM, n = 3-6. *P < 0.05; **P < 0.01; and ***P < 0.001. Similar results were obtained in three independent experiments. Fold change in (A) refers to t = 0. Control experiments indicated that hypotonicity per se had no effect (Appendix Fig S4).


Activation of LRRC8 channels by pro ‐ apoptotic stimuliA - CCisplatin ‐ induced iodide influx into WT ( black ■ ) , but not HAP1 − / − ( green ▼ ) HEK cells indicates VRAC halide current activation during apoptosis . Cells expressing an iodide ‐ sensitive YFP variant were exposed to 200 μM cisplatin for periods of 0 . 5 h ( A ) , 4 . 5 h ( B ) , or 8 . 5 h ( C ) before adding extracellular I − ( 50 mM final ) . The difference in slopes of YFPfluorescence quenching between control and cisplatin ‐ treated cells semiquantitatively reflects VRAC current activation . Note that increased YFP quenching with cisplatin preincubation is not due to large non ‐ specific leaks resulting from cell morbidity . Such leaks should lead to a fast component of YFP quenching in WT , but not HAP1 − / − cells after the pipetting artifact that immediately follows addition of iodide ( indicated by arrows ) . DSwelling ‐ induced iodide influx into WT ( black ■ ) and HAP1 − / − ( green ▼ ) HEK cells for comparison . Iodide ( 50 mM final ) was added in isotonic or hypotonic ( 230 mOsm final ) solution at the time indicated by arrow . E , FTime course of VRAC activation by 200 μM cisplatin ( E ) or 4 μM staurosporine ( F ) determined as in ( A - C ) . Averaged maximal slopes of YFP quenching from eight wells ( E ) or 16 wells ( F ) each were evaluated to estimate iodide influx rates . WT ( black ■ ) and HAP1 − / − ( green ▼ ) . Data are presented as mean ± SEM .

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

Activation of LRRC8 channels by pro‐apoptotic stimuliA-CCisplatin‐induced iodide influx into WT (black ■), but not HAP1−/− (green ▼) HEK cells indicates VRAC halide current activation during apoptosis. Cells expressing an iodide‐sensitive YFP variant were exposed to 200 μM cisplatin for periods of 0.5 h (A), 4.5 h (B), or 8.5 h (C) before adding extracellular I− (50 mM final). The difference in slopes of YFPfluorescence quenching between control and cisplatin‐treated cells semiquantitatively reflects VRAC current activation. Note that increased YFP quenching with cisplatin preincubation is not due to large non‐specific leaks resulting from cell morbidity. Such leaks should lead to a fast component of YFP quenching in WT, but not HAP1−/− cells after the pipetting artifact that immediately follows addition of iodide (indicated by arrows).DSwelling‐induced iodide influx into WT (black ■) and HAP1−/− (green ▼) HEK cells for comparison. Iodide (50 mM final) was added in isotonic or hypotonic (230 mOsm final) solution at the time indicated by arrow.E, FTime course of VRAC activation by 200 μM cisplatin (E) or 4 μM staurosporine (F) determined as in (A-C). Averaged maximal slopes of YFP quenching from eight wells (E) or 16 wells (F) each were evaluated to estimate iodide influx rates. WT (black ■) and HAP1−/− (green ▼). Data are presented as mean ± SEM.


Carboplatin uptake into control KBM7 cells and two LRRC8D ‐ deficient clones ( n = 6 ) .

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

Carboplatin uptake into control KBM7 cells and two LRRC8D‐deficient clones (n = 6).


Hypotonicity ‐ stimulated cisplatin uptake in HEK WT cells was selectively blocked by 100 μM carbenoxolone ( CBX ) , a non ‐ specific blocker of VRAC ( n = 3 ) .

O O O B-MEASURED_VAR 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

Hypotonicity‐stimulated cisplatin uptake in HEK WT cells was selectively blocked by 100 μM carbenoxolone (CBX), a non‐specific blocker of VRAC (n = 3).


Cisplatin uptake into HEK cells of indicated genotypes using 40 μM cisplatin under long ‐ term isotonic ( C ) or 200 μM cisplatin in short ‐ term hypo ‐ and isotonic ( D ) conditions as function of time ( n = 3 ) . Similar results were obtained in HCT116 cells ( Fig EV4 ) .

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

Cisplatin uptake into HEK cells of indicated genotypes using 40 μM cisplatin under long‐term isotonic (C) or 200 μM cisplatin in short‐term hypo‐ and isotonic (D) conditions as function of time (n = 3). Similar results were obtained in HCT116 cells (Fig EV4).


Cisplatin uptake ( 200 μM ) into HEK cells of indicated genotypes . LRRC8 ( B , C , E ) − / − and LRRC8 ( B , D , E ) − / − cells express only LRRC8A and LRRC8D , and LRRC8A and LRRC8C , respectively ( n = 3 for WT , 6 for LRRC8D − / − , 9 for LRRC8A − / − in E ; n = 3 in F ) .

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

Cisplatin uptake (200 μM) into HEK cells of indicated genotypes. LRRC8(B,C,E) −/− and LRRC8(B,D,E) −/− cells express only LRRC8A and LRRC8D, and LRRC8A and LRRC8C, respectively (n = 3 for WT, 6 for LRRC8D −/−, 9 for LRRC8A −/− in E; n = 3 in F).


Mean current densities of maximally activated ICl , vol at − 80 mV . The number of cells is indicated for each column .

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

Mean current densities of maximally activated ICl,vol at −80 mV. The number of cells is indicated for each column.


Ratio of LRRC8 ‐ dependent swelling ‐ activated cisplatin uptake ( 60 min ) to mean ICl , vol ( as in G ) as function of genotype . Data information : Data are presented as mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 ( for ( C , E ) compared to HAP1 − / − cells ) . Dotted lines in ( D ) and ( F ) highlight that there is no significant difference in isotonic 60 ‐ min uptake between the genotypes .

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

Ratio of LRRC8‐dependent swelling‐activated cisplatin uptake (60 min) to mean ICl,vol (as in G) as function of genotype.Data information: Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; and ***P < 0.001 (for (C, E) compared to HAP1−/− cells). Dotted lines in (D) and (F) highlight that there is no significant difference in isotonic 60‐min uptake between the genotypes.


Cisplatin - DMSO inhibition of ICl , vol depends on the LRRC8D subunitUpper panel , example current traces ( as in Fig 3A ) of fully activated ICl , vol in HEK cells exposed to hypotonic solution containing vehicle ( 0 . 3 % DMSO ) or 200 μM cisplatin in 0 . 3 % DMSO . Dashed lines indicate zero current . Lower panel , ICl , vol current densities ( at − 100 mV and 100 mV ) of WT HEK cells treated with different cisplatin concentrations . No effect of 200 μM cisplatin / DMSO on ICl , vol in LRRC8D − / − HEK cells . Data information : Data are presented as mean ± SEM ; the number of experiments is given for each bar ; * P < 0 . 05 ; * * P < 0 . 01 .

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

Cisplatin-DMSO inhibition of ICl,vol depends on the LRRC8D subunitUpper panel, example current traces (as in Fig 3A) of fully activated ICl,vol in HEK cells exposed to hypotonic solution containing vehicle (0.3% DMSO) or 200 μM cisplatin in 0.3% DMSO. Dashed lines indicate zero current. Lower panel, ICl,vol current densities (at −100 mV and 100 mV) of WT HEK cells treated with different cisplatin concentrations.No effect of 200 μM cisplatin/DMSO on ICl,vol in LRRC8D−/− HEK cells.Data information: Data are presented as mean ± SEM; the number of experiments is given for each bar; *P < 0.05; **P < 0.01.


Swelling ‐ induced efflux of 3 [ H ] ‐ taurine from WT and LRRC8D − / − HEK cells ( A , B ) and partial rescue by transient transfection of LRRC8D ( B ) . Rescue is incomplete due to low transfection / expression efficiency of LRRC8D ( Voss et al , 2014 ) .

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

Swelling‐induced efflux of 3[H]‐taurine from WT and LRRC8D −/− HEK cells (A, B) and partial rescue by transient transfection of LRRC8D (B). Rescue is incomplete due to low transfection/expression efficiency of LRRC8D (Voss et al, 2014).


C - ESwelling ‐ induced efflux of 3 [ H ] ‐ taurine from LRRC8 ( B , C , E ) − / − ( C ) , LRRC8 ( B , D , E ) − / − ( D ) , or LRRC8 ( B , C , D ) − / − ( E ) HEK cells compared to WT cells . FCisplatin ‐ induced taurine efflux ( over 30 min ) from WT and HAP1 − / − HEK cells after preincubation with 200 μM cisplatin for 4 or 12 h , or without cisplatin ( 0 h ) . Carbenoxolone ( CBX ; 100 μM ) blocks taurine efflux from WT cells treated for 12 h with cisplatin , excluding taurine flux through unspecific leaks . Data information : Data are presented as mean ± SEM . Dashed lines in ( A - E ) represent isotonic efflux from the same individual experiments . Red arrows , change to hypotonic solution . Each panel shows the average of two independent experiments ( n = 4 for each experiments , i . e . , n = 8 in total ) .

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

C-ESwelling‐induced efflux of 3[H]‐taurine from LRRC8(B,C,E) −/− (C), LRRC8(B,D,E) −/− (D), or LRRC8(B,C,D) −/− (E) HEK cells compared to WT cells.FCisplatin‐induced taurine efflux (over 30 min) from WT and HAP1−/−HEK cells after preincubation with 200 μM cisplatin for 4 or 12 h, or without cisplatin (0 h). Carbenoxolone (CBX; 100 μM) blocks taurine efflux from WT cells treated for 12 h with cisplatin, excluding taurine flux through unspecific leaks.Data information: Data are presented as mean ± SEM. Dashed lines in (A-E) represent isotonic efflux from the same individual experiments. Red arrows, change to hypotonic solution. Each panel shows the average of two independent experiments (n = 4 for each experiments, i.e., n = 8 in total).


( A ) Netrin - 1 ( NTN1 ) gene expression profiling from 6 adults control brains using microarray data from the Allen Human Brain Atlas .

O O O 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) Netrin-1 (NTN1) gene expression profiling from 6 adults control brains using microarray data from the Allen Human Brain Atlas.


( B ) Reporter gene expression in the midbrain ( substantia nigra ( SN ) and ventral tegmental area ( VTA ) ) of netrin - 1 + / lacZ + adult ( P60 ) mice incubated with S - Gal . ( Scale bar , 2000 µm ) . Two coronal sections of the mice midbrain are shown from the more anterior is shown in the upper panel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Reporter gene expression in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA)) of netrin-1+/lacZ+ adult (P60) mice incubated with S-Gal. (Scale bar, 2000 µm). Two coronal sections of the mice midbrain are shown from the more anterior is shown in the upper panel.


( C ) Netrin - 1 protein localization in the SN of adult rat . ( Scale bar , 200 µm ) . Upper panel , confocal image of the SN . Higher magnification of the SNpc ( bottoms panels ) . Netrin - 1 ( red ) , Tyrosine hydroxylase ( TH ) ( green ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Netrin-1 protein localization in the SN of adult rat. (Scale bar, 200 µm). Upper panel, confocal image of the SN. Higher magnification of the SNpc (bottoms panels). Netrin-1 (red), Tyrosine hydroxylase (TH) (green).


( D ) Localization of netrin - 1 protein in the adult rat striatum . ( Scale bar , 600µm ) . Left panel , confocal image of the rat striatum . Netrin - 1 ( red ) , Dopamine transporter ( DAT ) ( green ) . Right panels , higher magnification .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Localization of netrin-1 protein in the adult rat striatum. (Scale bar, 600µm). Left panel, confocal image of the rat striatum. Netrin-1 (red), Dopamine transporter (DAT) (green). Right panels, higher magnification.


( A ) Representative images of dopamine neurons ( TH - positive ) in the substantia nigra ( SN ) ( left panel ) and in the SN and striatum ( right panel top , and bottom respectively ) by immunofluorescence , six weeks after the intranigral injection of control ( CTL ) ( AAV6 - GFP ) or Cre ( AAV6 - GFP Cre ) adenovirus vectors in Netrin - 1fl / fl mice ( left panel : green - GFP , red - TH ; right panel : Cy - 5 - TH ) ( Scale bar , 1000 μm ) . Filled arrows indicate SN TH dopamine neurons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 dopamine neurons (TH-positive) in the substantia nigra (SN) (left panel) and in the SN and striatum (right panel top, and bottom respectively) by immunofluorescence, six weeks after the intranigral injection of control (CTL) (AAV6-GFP) or Cre (AAV6-GFP Cre) adenovirus vectors in Netrin-1fl/fl mice (left panel: green-GFP, red-TH; right panel: Cy-5-TH) (Scale bar, 1000 μm). Filled arrows indicate SN TH dopamine neurons.


( B ) Representative images of TH and netrin - 1 stainings by immunofluorescence ( scale bar , 2000 μm ) six weeks after the intranigral injection of CTL adenovirus vector ( left panel ) or Cre adenovirus vector ( right panel ) , in the midbrain ( substantia nigra ( SN ) and ventral tegmental area ( VTA ) of Netrin - 1fl / fl 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

(B) Representative images of TH and netrin-1 stainings by immunofluorescence (scale bar, 2000 μm) six weeks after the intranigral injection of CTL adenovirus vector (left panel) or Cre adenovirus vector (right panel), in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA) of Netrin-1fl/fl mice.


( C ) Quantification of fluorescence intensity of TH - positive neurons , in the substantia nigra ( SN ) ( left ) , and fibres , in the striatum ( right ) , six weeks after adenovirus vector injection , in Netrin - 1fl / fl mice . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 . N = 3 each 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of fluorescence intensity of TH-positive neurons, in the substantia nigra (SN) (left), and fibres, in the striatum (right), six weeks after adenovirus vector injection, in Netrin-1fl/fl mice. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; **P< 0.01. N=3 each group.


( D ) Terminal deoxynucleotidyl transferase ( TdT ) dUTP Nick - End Labelling ( TUNEL ) reactivity in the SN after Cre and control adenovirus vector injection , in Netrin - 1fl / fl mice ( Scale bar , 50μm ) . The apoptotic index is expressed as a percentage of TUNEL positive neurons out of the total number of TH - positive neurons . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * * P < 0 . 01 . N = 3 each 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labelling (TUNEL) reactivity in the SN after Cre and control adenovirus vector injection, in Netrin-1fl/fl mice (Scale bar, 50μm). The apoptotic index is expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. **P< 0.01. N=3 each group.


( E ) Motor behavior assays at six weeks after treatment . N = 8 animals / group . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * P < 0 .

O O O O O O O O O O O O O O O O O O O O O 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) Motor behavior assays at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05


( F ) Immunoblot of SN lysates from netrin - 1 wild type ( WT ) and netrin - 1 conditional KO mice ( left upper panel ) . Densitometry band quantification of cleaved receptor over full length receptor ratio ( lower right panel ) . We randomly selected 2 mice from each group for immunoblot analysis . N = 3 independent experiments . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * P < 0 . 05 ; P * * < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot of SN lysates from netrin-1 wild type (WT) and netrin-1 conditional KO mice (left upper panel). Densitometry band quantification of cleaved receptor over full length receptor ratio (lower right panel). We randomly selected 2 mice from each group for immunoblot analysis. N=3 independent experiments. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01.


( A ) Representative images of TH + dopamine neurons and fibers staining by immunofluorescence , in the SN and the striatum ( Str ) 6 weeks after CTL ( AAV6 - GFP ) ; Cre ( AAV6 - GFP Cre ) ; sh DCC ( AAV6 - GFP Cre + Sh - DCC ) and shUNC5B ( AAV6 GFP Cre + Sh - UNC5B ) injection into the SN of Netrin - 1fl / fl mice . ( Scale bar , 2000 µm ) . ( B ) Quantification of TH fluorescent intensity in the SN ( upper bar graph ) and striatum ( Str ) of Netrin - 1fl / fl mice ( lower bar graph ) . Data are shown as mean + SEM . Statistical significance was determined by an unpaired t - test . N = 3 each group . Tukey post hoc analysis after one - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , N . S . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 TH+ dopamine neurons and fibers staining by immunofluorescence, in the SN and the striatum (Str) 6 weeks after CTL (AAV6-GFP); Cre (AAV6-GFP Cre); sh DCC (AAV6-GFP Cre + Sh-DCC) and shUNC5B (AAV6 GFP Cre + Sh-UNC5B) injection into the SN of Netrin-1fl/fl mice. (Scale bar, 2000 µm). (B) Quantification of TH fluorescent intensity in the SN (upper bar graph) and striatum (Str) of Netrin-1fl/fl mice (lower bar graph). Data are shown as mean + SEM. Statistical significance was determined by an unpaired t-test. N = 3 each group. Tukey post hoc analysis after one-way ANOVA *P < 0.05, **P <0.01, N.S., not significant.