D . of Fbxl17 mRNA in PC3 DAOY transfected with a non - targeting siRNA ( Control ) or two siRNAs to Fbxl17 ( 1 ) ( 2 ) . were serum starved for 24 hours in serum - reduced medium treated with SAG ( 100 nM ) for 24 hours prior collection .

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D. Quantification of Fbxl17 mRNA levels in PC3 and DAOY transfected with a non-targeting siRNA (Control) or two siRNAs to Fbxl17 (1) and (2). Cells were serum starved for 24 hours in serum-reduced medium and treated with SAG (100 nM) for 24 hours prior collection.


E . Sufu in PC3 DAOY treated as in D . GAPDH was used as a loading control . Representative image of three independent experiments is shown .

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E. Sufu protein levels in PC3 and DAOY cells treated as in D. GAPDH was used as a loading control. Representative image of three independent experiments is shown.


F . Sufu in PC3 DAOY infected with an empty backbone retrovirus ( EV ) or a retrovirus expressing Myc - tagged Fbxl17 . Representative image from three independent experiments is shown .

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

F. Sufu protein levels in PC3 and DAOY cells infected with an empty backbone retrovirus (EV) or a retrovirus expressing Myc-tagged Fbxl17. Representative image from three independent experiments is shown.


G . Detection of Sufu following cycloheximide ( CHX ) treatment for indicated hours , Fbxl17 depletion using two different siRNAs . ( Hrs = hours ) . Representative image of two independent experiments is

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G. Detection of Sufu protein levels following cycloheximide (CHX) treatment for the indicated hours, and Fbxl17 depletion using two different siRNAs. (Hrs=hours). Representative image of two independent experiments is shown


A . Detection of polyubiquitylated species of endogenous Sufu co - immunopurified from DAOY transfected with Myc - tagged ubiquitin ( Ub ) either in presence of non - targeting siRNA ( Control ) or two siRNAs against Fbxl17 ( 1 ) ( 2 ) . MG132 ( 10µM ) was added in samples .

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

A. Detection of polyubiquitylated species of endogenous Sufu co-immunopurified from DAOY cells transfected with Myc-tagged ubiquitin (Ub) either in the presence of non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2). MG132 (10µM) was added in all samples.


B . Detection of polyubiquitylated species of Sufu upon co - transfection of HEK293T with HA - tagged Sufu Myc - tagged ubiquitin ( Ub ) either in presence of non - targeting siRNA ( Control ) or two siRNAs against Fbxl17 ( 1 ) ( 2 ) . MG132 ( 10µM ) was added in samples .

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B. Detection of polyubiquitylated species of Sufu upon co-transfection of HEK293T cells with HA-tagged Sufu and Myc-tagged ubiquitin (Ub) either in the presence of non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2). MG132 (10µM) was added in all samples.


C . Fbxl17 mRNA relative in DAOY HEK293T upon Fbxl17 depletion with either a non - targeting siRNA ( Control ) or two siRNAs to Fbxl17 ( 1 ) ( 2 ) .

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C. Fbxl17 mRNA relative levels in DAOY and HEK293T cells upon Fbxl17 depletion with either a non-targeting siRNA (Control) or two siRNAs to Fbxl17 (1) and (2).


D . Detection of ubiquitylated Sufuco - immunoprecipitated from HEK293T co - transfected with HA - tagged Sufu , Myc - ubiquitin ( Ub ) along with either Flag - tagged Fbxl17 wild type ( WT ) or a mutant lacking F - box domain ( Fbxl17ΔF ) . MG132 ( 10µM ) was added in samples .

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

D. Detection of ubiquitylated Sufuco-immunoprecipitated from HEK293T cells co-transfected with HA-tagged Sufu, Myc-ubiquitin (Ub) along with either Flag-tagged Fbxl17 wild type (WT) or a mutant lacking the F-box domain (Fbxl17ΔF). MG132 (10µM) was added in all samples.


E . Detection of ubiquitylated Sufuco - immunoprecipitated from HEK293T co - transfected with Myc - tagged ubiquitin ( Ub ) , Flag - tagged Fbxl17 HA - tagged Sufu WT or Sufu mutant K257R . MG132 ( 10µM ) was added in samples .

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

E. Detection of ubiquitylated Sufuco-immunoprecipitated from HEK293T cells co-transfected with Myc-tagged ubiquitin (Ub), Flag-tagged Fbxl17 and HA-tagged Sufu WT or Sufu mutant K257R. MG132 (10µM) was added in all samples.


A . Detection of Myc - tagged Fbxl17 after immunoprecipitation of HA - tagged Sufu WT or Sufu S342 / 6A S342 / 6D , as indicated . HEK293T were treated with MLN4924 ( 2 M ) for 5 hours prior collection .

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

A. Detection of Myc-tagged Fbxl17 after immunoprecipitation of HA-tagged Sufu WT or Sufu S342/6A and S342/6D, as indicated. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.


B . Detection of Flag - tagged Gli1 Myc - tagged Fbxl17 binding to immunoprecipitated HA - tagged Sufu Wild - Type ( WT ) or to Sufu S352F . An Empty Vector ( EV ) was used as a negative control . HEK293T were treated with MLN4924 ( 2 M ) for 5 hours prior collection .

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B. Detection of Flag-tagged Gli1 and Myc-tagged Fbxl17 binding to immunoprecipitated HA-tagged Sufu Wild-Type (WT) or to Sufu S352F. An Empty Vector (EV) was used as a negative control. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.


C . Detection of phosphorylated Sufu on S352 / T353 after immunoprecipitation of HA - tagged Sufu WT Sufu 351 - 353 AAA , as indicated .

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C. Detection of phosphorylated Sufu on S352/T353 after immunoprecipitation of HA-tagged Sufu WT and Sufu 351-353 AAA, as indicated.


D . Detection of Myc - tagged Fbxl17 binding to Sufu full ( FL ) or to following Sufu peptides : Sufu 340 - 360 ( APSRKDSLESDSSTAIIPHEL ) ; Sufu 340 - 360 [ S352P / T353P ] ( phosphorylated on residues S352 T353 ) ; Sufu 351 - 372 ( SSTAIIPHELIRTRQLESVHLK ) .

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D. Detection of Myc-tagged Fbxl17 binding to Sufu full length (FL) or to the following Sufu peptides: Sufu 340-360(APSRKDSLESDSSTAIIPHEL); Sufu 340-360 [S352P/T353P] (phosphorylated on the residues S352 and T353); Sufu 351-372 (SSTAIIPHELIRTRQLESVHLK).


E . Detection of HA - tagged Sufu binding to a Myc - tagged Fbxl17 construct encompassing residues 318 to 701 ( corresponding to F - box motif C - terminus region ) assessed by in vitro binding . Both were synthetized in vitro using a T7 - coupled reticulocyte lysate system .

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

E. Detection of HA-tagged Sufu binding to a Myc-tagged Fbxl17 construct encompassing the residues 318 to 701 (corresponding to the F-box motif and C-terminus region) assessed by in vitro binding assay. Both proteins were synthetized in vitro using a T7-coupled reticulocyte lysate system.


F . Detection of ubiquitylated Sufu immunoprecipitated from HEK293T co - transfected with HA - tagged ubiquitin ( Ub ) , Myc - tagged Fbxl17 Flag - tagged Gli1 . MG132 ( 10µM ) was added in samples .

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

F. Detection of ubiquitylated Sufu immunoprecipitated from HEK293T cells co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged Fbxl17 and Flag-tagged Gli1. MG132 (10µM) was added in all samples.


G . Detection of Flag - tagged Gli1 binding to Myc - tagged Fbxl17 co - immunoprecipitated from HEK293T transfected with a non - targeting siRNA ( Control ) or two siRNA to Sufu ( 1 ) ( 2 ) , as indicated .

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

G. Detection of Flag-tagged Gli1 binding to Myc-tagged Fbxl17 co-immunoprecipitated from HEK293T cells transfected with a non-targeting siRNA (Control) or two siRNA to Sufu (1) and (2), as indicated.


A . Sufu in mouse embryonic fibroblasts ( MEFs ) Ptch1 + / + , Ptch1 + / - Ptch1 - / - transfected with a non - targeting siRNA ( Control ) or two siRNAs targeting mouse Fbxl17 ( 1 ) ( 2 ) .

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A. Sufu protein levels in mouse embryonic fibroblasts (MEFs) Ptch1+/+, Ptch1+/- and Ptch1-/- transfected with a non-targeting siRNA (Control) or two siRNAs targeting mouse Fbxl17 (1) and (2).


B . of Fbxl17 mRNA in MEFs Ptch1 + / + , Ptch1 + / - Ptch1 - / - transfected as in A .

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B. Quantification of Fbxl17 mRNA levels in MEFs Ptch1+/+, Ptch1+/- and Ptch1-/- transfected as in A.


C . of Gli1 mRNA in MEFs Ptch1 + / + Ptch1 - / - upon Fbxl17 depletion using two different siRNAs . ( mean ± from 3 independent experiments , * * * p < 0 . 0005 ) .

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C. Analysis of Gli1 mRNA levels in MEFs Ptch1+/+ and Ptch1-/- upon Fbxl17 depletion using two different siRNAs. (mean ± SEM from 3 independent experiments, ***p<0.0005).


D . of Bcl2 mRNA in MEFs Ptch1 + / + Ptch1 - / - upon Fbxl17 depletion using two different siRNAs . ( mean ± from 3 independent experiments , * * p < 0 . 005 ; * * * p < 0 . 0005 ) .

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D. Analysis of Bcl2 mRNA levels in MEFs Ptch1+/+ and Ptch1-/- upon Fbxl17 depletion using two different siRNAs. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).


E . Detection of Flag - tagged Gli1 binding to HA - tagged Sufu in MEFs Ptch1 - / - upon expression of Myc - tagged Fbxl17 , or Fbxl17 depletion by two siRNAs . For each condition , 200 µg of whole lysate was immunoprecipitated .

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E. Detection of Flag-tagged Gli1 binding to HA-tagged Sufu in MEFs Ptch1-/- cells upon expression of Myc-tagged Fbxl17, or Fbxl17 depletion by two siRNAs. For each condition, 200 µg of whole cell lysate was immunoprecipitated.


F . Immunostaining of Fbxl17 using anti - Myc antibody in Ptch1 - / - MEFs stably transduced with a pBABE vector expressing Fbxl17 tagged with Myc either at N - terminus [ Myc ( N ) - Fbxl17 ] or at C - terminus [ Myc ( C ) - Fbxl17 ] . Scale bars : 20 m .

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F. Immunostaining of Fbxl17 using anti-Myc antibody in Ptch1-/- MEFs stably transduced with a pBABE vector expressing Fbxl17 tagged with Myc either at the N-terminus [Myc(N)-Fbxl17] or at the C-terminus [Myc(C)-Fbxl17]. Scale bars: 20 m.


G . Detection of Fbxl17 using anti - Myc antibody in Ptch1 - / - MEFs stably expressing Myc ( N ) - Fbxl17 ( Fbxl17 tagged at N - terminus ) Myc ( C ) - Fbxl17 ( Fbxl17 tagged at C - terminus ) .

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G. Detection of Fbxl17 using anti-Myc antibody in Ptch1-/- MEFs stably expressing Myc(N)-Fbxl17 (Fbxl17 tagged at the N-terminus) and Myc(C)-Fbxl17 (Fbxl17 tagged at the C-terminus).


H . Detection of HA - tagged Fbxl17 binding to endogenous Sufu immunoprecipitated from extracts from Ptch1 - / - MEFs . Asterisk ( * ) indicates a non specific band . Identification of fractions was performed by Lamin A / C GAPDH detection .

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

H. Detection of HA-tagged Fbxl17 binding to endogenous Sufu immunoprecipitated from cytoplasmic and nuclear extracts from Ptch1-/- MEFs. Asterisk (*) indicates a non specific band. Identification of cytoplasmic and nuclear fractions was performed by Lamin A/C and GAPDH detection.


A . proliferation of DAOY upon Fbxl17 depletion using a non - targeting siRNA ( Control ) , two siRNAs against Fbxl17 ( 1 ) ( 2 ) or upon reintroduction of a Myc - tagged Fbxl17 construct in Fbxl17 - depleted DAOY using siRNA Fbxl17 ( 2 ) ( mean ± from 3 independent experiments , * * p < 0 . 005 ; * * * p < 0 . 0005 ) .

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

A. Cell proliferation of DAOY cells upon Fbxl17 depletion using a non-targeting siRNA (Control), two siRNAs against Fbxl17 (1) and (2) or upon reintroduction of a Myc-tagged Fbxl17 construct in Fbxl17-depleted DAOY cells using siRNA Fbxl17 (2) (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).


B . of Fbxl17 mRNA in DAOY transfected with a non - targeting siRNA ( Control ) or two siRNAs against Fbxl17 ( 1 ) ( 2 ) ( mean ± from 3 independent experiments , * * p < 0 . 005 ) .

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

B. Quantification of Fbxl17 mRNA levels in DAOY cells transfected with a non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2) (mean ± SEM from 3 independent experiments, **p<0.005).


C . Sufu in DAOY treated as in A . Representative image of three independent experiments is shown .

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C. Sufu protein levels in DAOY cells treated as in A. Representative image of three independent experiments is shown.


D . proliferation of DAOY transfected with non - targeting siRNA ( Control ) , siRNA against Fbxl17 or a combination of siRNA targeting Fbxl17 Sufu . ( mean ± from 3 independent experiments , * * p < 0 . 005 ; * * * p < 0 . 0005 ) .

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

D. Cell proliferation of DAOY cells transfected with non-targeting siRNA (Control), siRNA against Fbxl17 or a combination of siRNA targeting Fbxl17 and Sufu. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).


E . of Fbxl17 mRNA in DAOY treated as in D mean ± from 3 independent experiments , * * * p < 0 . 0005 ) .

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

E. Quantification of Fbxl17 mRNA levels in DAOY cells treated as in D mean ± SEM from 3 independent experiments, ***p<0.0005).


F . Sufu in DAOY treated as in D . Representative image of three independent experiments is shown .

F. Sufu protein levels in DAOY cells treated as in D. Representative image of three independent experiments is shown.


A . of Fbxl17 mRNA in DAOY transfected with control shRNA or shRNA targeting Fbxl17 . ( mean ± from 3 independent experiments , * * * p < 0 . 0005 ) .

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A. Quantification of Fbxl17 mRNA levels in DAOY cells transfected with control shRNA or shRNA targeting Fbxl17. (mean ± SEM from 3 independent experiments, ***p<0.0005).


B . Detection of Sufu in DAOY transfected with control shRNA or shRNA Fbxl17 . Representative image of three independent experiments is shown .

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B. Detection of Sufu protein levels in DAOY cells transfected with control shRNA or shRNA Fbxl17. Representative image of three independent experiments is shown.


C . of Gli1 mRNA in DAOY transfected with control shRNA or shRNA Fbxl17 . ( mean ± from 3 independent experiments , * * p < 0 . 005 ) .

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C. Quantification of Gli1 mRNA levels in DAOY cells transfected with control shRNA or shRNA Fbxl17. (mean ± SEM from 3 independent experiments, **p<0.005).


D . T2 - weighted magnetic resonance imaging ( MRI ) showing tumour development in rats injected with DAOY transfected with control shRNA or shRNA against Fbxl17 . One representative image for each condition , at 10 is shown . Scale bar : 5mm . E . Graph showing of T2 hyperintensity ( a surrogate marker of tumourgrowth ) between 4 10 post - tumour induction . were injected with DAOY stably expressing control shRNA or shRNA against Fbxl17 ( 10 , 000 / 1µL ) . ( mean ± ; n = 5 ; two - way ANOVA , followed by unpaired t - , * p < 0 . 05 , * * p < 0 . 01 ) .

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

D. T2- weighted magnetic resonance imaging (MRI) showing tumour development in rats injected with DAOY cells transfected with control shRNA or shRNA against Fbxl17. One representative image for each condition, at 10 weeks is shown. Scale bar: 5mm. E. Graph showing area of T2 hyperintensity (a surrogate marker of tumourgrowth) between 4 and 10 weeks post-tumour induction. Animals were injected with DAOY cells stably expressing control shRNA or shRNA against Fbxl17 (10,000 cells/1µL). (mean ± SEM; n = 5; two-way ANOVA, followed by unpaired t-test, *p<0.05, **p<0.01).


F . Representative immunohistochemistry image of Vimentin . Scale bar : 1 mm . G . of tumour growth ( vimentin ) in rats injected with DAOY transfected with either control shRNA or shRNA against Fbxl17 ( mean ± ; n = 5 ; unpaired t - , * p < 0 . 05 ) .

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F. Representative immunohistochemistry image of Vimentin staining. Scale bar: 1 mm.G. Quantification of tumour growth (vimentin staining) in rats injected with DAOY cells transfected with either control shRNA or shRNA against Fbxl17 (mean ± SEM; n = 5; unpaired t-test, *p<0.05).


H . Representative immunohistochemistry image of Ki67 . Scale bar : 0 . 5 mm / 0 . 1 mm . I . of proliferative index ( Ki67 ) in rats injected with DAOY transfected with either control shRNA or shRNA against Fbxl17 ( mean ± ; n = 5 ; unpaired t - , * p < 0 . 05 ) .

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

H. Representative immunohistochemistry image of Ki67 staining. Scale bar: 0.5 mm/0.1 mm.I. Quantification of proliferative index (Ki67 staining) in rats injected with DAOY cells transfected with either control shRNA or shRNA against Fbxl17 (mean ± SEM; n = 5; unpaired t-test, *p<0.05).


A - C . mRNA expression of Sufu , Gli1 Fbxl17 detected in 285 medulloblastoma samples ( GEO accession : GSE37382 ) , stratified by subtype . Level of significance ( p ) for Kruskal - Wallis ( KW ) rank is p < 10 - 8 . using one - way anova gave comparable results .

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A-C. mRNA expression of Sufu, Gli1 and Fbxl17 detected in 285 medulloblastoma samples (GEO accession: GSE37382), stratified by tumor subtype. Level of significance (p) for Kruskal-Wallis (KW) rank test is p<10-8. Analysis using one-way anova gave comparable results.


D . Expression of Gli1 Fbxl17 in 285 is shown colored by subgroup ; level of significance ( p ) for Kruskal - Wallis ( KW ) rank is p < 10 - 8 , Spearman ' s rank correlation ( R = 0 . 5640 ) is indicated .

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D. Expression of Gli1 and Fbxl17 in the 285 patients is shown colored by subgroup; level of significance (p) for Kruskal-Wallis (KW) rank test is p<10-8, and Spearman's rank correlation (R=0.5640) is indicated.


E . Detection of HA - tagged Sufu wild type ( WT ) HA - tagged Sufu S352F in NIH3T3 after cycloheximide ( CHX ) treatment . Where indicated , were transfected with a non - targeting siRNA ( Control ) or two siRNA to Fbxl17 ( 1 ) ( 2 ) .

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E. Detection of HA-tagged Sufu wild type (WT) and HA-tagged Sufu S352F protein levels in NIH3T3 cells after cycloheximide (CHX) treatment. Where indicated, cells were transfected with a non- targeting siRNA (Control) or two siRNA to Fbxl17 (1) and (2).


F . Assessment of Hh pathway activation using Gli1 - luciferase reporter in MEFs Sufu - / - upon reintroduction of HA - tagged Sufu WT or Sufu mutant S352F using a retroviral system . ( mean ± from 3 independent experiments , * * p < 0 . 005 ; * * * p < 0 . 0005 ) .

O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Assessment of Hh pathway activation using Gli1-luciferase reporter assay in MEFs Sufu-/- upon reintroduction of HA-tagged Sufu WT or Sufu mutant S352F using a retroviral system. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).


T2 - weighed brain MRIs of A ( D1 - 3 ) at 28 months of B at ( D4 - 6 ) at 16 months of , showing global brain atrophy as well as periventricular deep matter hyperintensities .

O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-DISEASE O O O B-TISSUE I-TISSUE I-TISSUE O O

T2-weighed brain MRIs of patient A (D1-3) at 28 months of age and patient B at (D4-6) at 16 months of age, showing global brain atrophy as well as periventricular and deep white matter hyperintensities.


Quantifications of HOPS / CORVET subunits in fibroblast lysates ( top ) representative immunoblots ( bottom ) , analyzed as in ( C ) . ( E ) VPS33A , ( F ) VPS11 , ( G ) VPS18 .

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

Quantifications of HOPS/CORVET subunits in fibroblast lysates (top) and representative immunoblots (bottom), analyzed as in (C). (E) VPS33A, (F) VPS11, (G) VPS18.


Immunoblots ( left ) quantifications normalized to control ( right ) of VPS16 , VPS33A VPS11 in lysates from fibroblasts transduced with a lentivirus to express VPS16 or control ( empty vector ) .

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

Immunoblots (left) and quantifications normalized to control (right) of VPS16, VPS33A and VPS11 in lysates from fibroblasts transduced with a lentivirus to express VPS16 or control (empty vector).


of early endosome marker EEA1 in fibroblasts transduced with control or VPS16 - expressing lentivirus by confocal microscopy . ( A ) Representative confocal . Scale bar , 20 μm . ( B ) of EEA1 - stained number intensities ( ca 80 analyzed for each of n = 3 biological replicates ) .

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

Analysis of the early endosome marker EEA1 in fibroblasts transduced with control or VPS16-expressing lentivirus by confocal microscopy. (A) Representative confocal micrographs. Scale bar, 20 μm. (B) Quantification of EEA1-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates).


of intracellular Transferrin trafficking . Fibroblasts fed with fluorescently labeled Transferrin were co - stained with LysoTracker ( G ) or an antibody against LAMP2 ( H ) . Representative ( left ) of fibroblasts transduced with control , with magnified areas demonstrating co - localized ( arrowheads ) . Scale bars 10 μm or 2 μm ( inserts ) . Quantifications ( right ) of co - localization between Transferrin LysoTracker or LAMP2 , respectively , expressed as Pearson correlation coefficients . Colored horizontal bars indicate median values whiskers 5 95 percentiles ( n = 10 optical sections from 3 independent experiments ) .

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

Analysis of intracellular Transferrin trafficking. Fibroblasts fed with fluorescently labeled Transferrin were co-stained with LysoTracker (G) or an antibody against LAMP2 (H). Representative images (left) of fibroblasts transduced with control virus, with magnified areas demonstrating co-localized puncta (arrowheads). Scale bars 10 μm or 2 μm (inserts). Quantifications (right) of the co-localization between Transferrin and LysoTracker or LAMP2, respectively, expressed as Pearson correlation coefficients. Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles (n=10 optical sections from 3 independent experiments).


Confocal of fibroblasts transduced with control or VPS16 - expressing lentivirus labeled with LysoTracker . Scale bar , 20 μm . of LysoTracker - stained number intensities ( ca 80 analyzed for each of n = 3 biological replicates ) .

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

Confocal micrographs of fibroblasts transduced with control or VPS16-expressing lentivirus and labeled with LysoTracker. Scale bar, 20 μm. Quantification of LysoTracker-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates).


Representative immunoblots ( bottom ) summary quantifications ( top ) of of LAMP1 ( C ) LAMP2 ( D ) in fibroblasts , normalized to of actin ( ACTB ) expressed as % of controls ( n = 3 biological replicates ) . of lysosomal Cathepsin D ( CtsD ) processing by immunoblotting . Immunoblot to detect CtsD isoforms ( left ) ( right ) of processing , calculated as of CtsD divided by sum of isoforms ( n = 3 biological replicates ) .

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

Representative immunoblots (bottom) and summary quantifications (top) of the levels of LAMP1 (C) and LAMP2 (D) in fibroblasts, normalized to levels of actin (ACTB) and expressed as % of controls (n=3 biological replicates). Analysis of lysosomal Cathepsin D (CtsD) processing by immunoblotting. Immunoblot to detect CtsD isoforms (left) and quantification (right) of processing, calculated as the percentage of CtsD divided by the sum of all isoforms (n=3 biological replicates).


of autophagosome numbers in fibroblasts transfected to express mRFP - GFP - LC3 . ( F ) Representative confocal ( optical sections ) of fibroblasts imaged live under basal or serum - starved conditions . Scale bar , 20 μm .

O B-SUBCELLULAR O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O

Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. (F) Representative confocal micrographs (optical sections) of fibroblasts imaged live under basal or serum-starved conditions. Scale bar, 20 μm.


of autophagosome numbers in fibroblasts transfected to express mRFP - GFP - LC3 . of autophagosomes , defined as green + red ( G ) autophago - lysosomes , calculated as number of red ( but not green ) divided by total number of . Colored horizontal bars denote median values whiskers 5 95 percentiles ( n = 50 - 113 from a total of 3 independent experiments ; for exact values see Appendix Table S1 ) .

O B-SUBCELLULAR O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. Quantification of autophagosomes, defined as green + red puncta (G) and autophago-lysosomes, calculated as the number of red (but not green) puncta divided by the total number of puncta. Colored horizontal bars denote median values and whiskers 5 and 95 percentiles (n=50-113 cells from a total of 3 independent experiments; for exact values see Appendix Table S1).


Myelination , as reported using Tg ( mbp : gfp - caax ) transgenic , is significantly reduced in vps16 crispants . yellow box indicates region assessed in D . Scale bar 100 μm . Bar graphs represent data as mean ± . Statistical comparisons by Mann - Whitney U ( n = 6 ) . * * p < 0 . 01 .

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

Myelination, as reported using the Tg(mbp:gfp-caax) transgenic, is significantly reduced in vps16 crispants. The yellow box indicates the region assessed in D. Scale bar 100 μm. Bar graphs represent data as mean ±SEM. Statistical comparisons by Mann-Whitney U tests (n=6). ** p < 0.01.


LysoTracker signal colocalizes with astroglial reporter slc1a2b : citrine does not appear to overlap with pan - neuronal reporter elavl3 : GCaMP5G .

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LysoTracker signal colocalizes with the astroglial reporter slc1a2b:citrine and does not appear to overlap with the pan-neuronal reporter elavl3:GCaMP5G.


b . In vitro activity . Left y - axis : 13C2 - oxalate in mM , right y - axis : 13C - formate in mM , x - axis : time in minutes . SYNB8802 ( blue , circles , solid : oxalate , dotted : formate ) control EcN ( pink , triangle , solid : oxalate , dotted : formate ) . Three biological replicates were run plotted separately .

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

b. In vitro activity. Left y-axis: 13C2-oxalate in mM, right y-axis: 13C-formate in mM, x-axis: time in minutes. SYNB8802 (blue, circles, solid line: oxalate, dotted line: formate) The control EcN (pink, triangle, solid line: oxalate, dotted line: formate). Three biological replicates were run and plotted separately.


c . In vitro simulation ( IVS ) . Left y - axis : Rate of oxalate degradation in μmol / h / 109 . X - axis = time in hours . Left X - axis 0 - 4 hours , Right X - axis 6 - 48 h . Three biological replicates were run plotted separately . Data points in orange box represent incubation in simulated gastric fluid ( SGF ) . Data points in blue box represent incubation in simulated intestinal fluid ( SIF ) . Data points in pink box represent incubation in simulated colonic fluid ( SCF ) .

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

c. In vitro simulation (IVS). Left y-axis: Rate of oxalate degradation in μmol/h/109 cells. X-axis = time in hours. Left X-axis 0 - 4 hours, Right X-axis 6 - 48 h. Three biological replicates were run and plotted separately. Data points in the orange box represent incubation in simulated gastric fluid (SGF). Data points in the light blue box represent incubation in simulated intestinal fluid (SIF). Data points in the pink box represent incubation in simulated colonic fluid (SCF).


b . Acute mouse study of UOx lowering with SYNB8802 . y - axis shows urinary 13 C2 - oxalate normalized to creatine . x - axis shows two groups , EcN ( control , pink ) vs SYNB8802AbxR ( blue ; n = 15 mice per group ) . dots represent each metabolic cage ( n = 3 mice per metabolic cage ) . Error bars are calculated as . ( unpaired t - with Welch ' s correction , * * * * p < 0 . 0001 )

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

b. Acute mouse study of UOx lowering with SYNB8802. The y-axis shows the urinary 13 C2-oxalate normalized to creatine. The x-axis shows the two groups, EcN (control, pink) vs SYNB8802AbxR (light blue; n = 15 mice per group). Individual dots represent each metabolic cage (n = 3 mice per metabolic cage). Error bars are calculated as SEM. (unpaired t-test with Welch's correction, **** p < 0.0001)


d . Nonhuman primate model development study . y - axis shows urinary oxalate normalized to creatine . X - axis shows baseline urinary oxalate after overnight fast , urinary oxalate measured 6 hours post administration of either a spinach preparation ( triangle , pink ) or water ( circle , blue ) ( n = 6 for each group ) . dots represent . Error bars are calculated as . ( Two - way ANOVA followed by Sidak ' s multiple comparison , * * * * p < 0 . 0001 ) .

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

d. Nonhuman primate model development study. The y-axis shows urinary oxalate normalized to creatine. X-axis shows baseline urinary oxalate levels after overnight fast, and urinary oxalate measured 6 hours post administration of either a spinach preparation (triangle, pink) or water (circle, blue) (n=6 for each group). Individual dots represent individual animals. Error bars are calculated as SEM. (Two-way ANOVA followed by Sidak's multiple comparison analysis, **** p < 0.0001).


f . Urinary recovery of oxalate in nonhuman primates . y - axis shows change in urinary oxalate from vehicle control . x - axis shows vehicle ( control , grey ) increasing doses of SYNB8802AbxR ( n = 24 for vehicle , n = 6 for treatment groups ) . dots represent normalized urinary oxalate for each . Error bars are calculated as . ( One - way ANOVA followed by Tukey ' s multiple comparison , * p < 0 . 05 )

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

f. Urinary recovery of oxalate in nonhuman primates. The y-axis shows change in urinary oxalate from vehicle control. The x-axis shows vehicle (control, grey) and increasing doses of SYNB8802AbxR (n = 24 for vehicle, n = 6 for treatment groups). Individual dots represent normalized urinary oxalate for each individual animal. Error bars are calculated as SEM. (One-way ANOVA followed by Tukey's multiple comparison analysis, * p < 0.05)


g . Urinary recovery of 13C2 oxalate in nonhuman primates . y - axis shows change in urinary 13C2 - oxalate from vehicle control . x - axis shows vehicle ( control , grey ) increasing doses of SYNB8802AbxR ( n = 24 for vehicle , n = 6 for treatmentgroups ) . dots represent normalized urinary 13C2 - oxalate for each . Error bars are calculated as . ( One - way ANOVA followed by Tukey ' s multiple comparison , * * p < 0 . 01 )

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

g. Urinary recovery of 13C2 oxalate in nonhuman primates. The y-axis shows change in urinary 13C2-oxalate from vehicle control. The x-axis shows vehicle (control, grey) and increasing doses of SYNB8802AbxR (n = 24 for vehicle, n = 6 for treatmentgroups). Individual dots represent normalized urinary 13C2-oxalate for each individual animal. Error bars are calculated as SEM. (One-way ANOVA followed by Tukey's multiple comparison analysis, ** p < 0.01)


( B ) Time constants of decay , τdecay , of STC isolated pharmacologically in WT FHM2 KI mice . Top , superimposed representative traces of inward current evoked in an astrocyte ( held at - 90 mV ) by a single stimulation ( indicated by arrow ) in a WT slice , before ( trace a ) after ( trace b ) application of a saturating of GluT inhibitor TFB - TBOA ( TBOA ) . STC was obtained by subtracting residual current remaining in presence of TBOA from total inward current ( trace a - b ) ; decay of STC was best fitted by a single exponential function with τdecay = 6 . 53 ms ( in red ) . bar plot shows average values of τdecay of STC isolated pharmacologically in cortical slices ( n = 13 ) from P22 - 23 WT ( N = 7 ) KI mice ( n = 9 ; N = 3 ) . STC τdecay is 17 % higher in KI compared to WT astrocytes ( unpaired t - : P = 0 . 003 ) . Hereafter , n indicates number of slices N indicates number of mice .

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

(B) Time constants of decay, τdecay, of the STC isolated pharmacologically in WT and FHM2 KI mice. Top, superimposed representative traces of the inward current evoked in an astrocyte (held at -90 mV) by a single pulse stimulation (indicated by the arrow) in a WT slice, before (trace a) and after (trace b) application of a saturating concentration of the GluT inhibitor TFB-TBOA (TBOA). The STC was obtained by subtracting the residual current remaining in the presence of TBOA from the total inward current (trace a-b); the decay of the STC was best fitted by a single exponential function with τdecay = 6.53 ms (in red). The bar plot shows the average values of τdecay of the STC isolated pharmacologically in cortical slices (n=13) from P22-23 WT (N=7) and KI mice (n=9; N=3). STC τdecay is 17% higher in KI compared to WT astrocytes (unpaired t-test: P = 0.003). Hereafter, n indicates the number of slices and N indicates the number of mice.


( C ) τdecay of STC isolated using an exponential waveform approximating average TBOA - insensitive current in WT KI mice . Top trace , average normalized TBOA - insensitive current obtained by pooling normalized TBOA - insensitive currents recorded in 16 WT KI . This current was best fitted by an exponentially rising function ( 1 - exp ( - t / τrise ) ) with τrise = 2 . 35 ms ( in green ) . STC was obtained by subtracting from total current elicited in astrocyte ( a : same representative trace as in ( B ) ) exponential function A ( 1 - exp ( - t / τrise ) ) with τrise = 2 . 35 ms A equal to maximal current measured in astrocyte at about 60 ms after stimulation ( trace c ) ; decay of STC ( trace a - c ) was best fitted by a single exponential function with τdecay = 6 . 49 ms ( in red ) . bar plot shows average values of τdecay of STC isolated as shown in top panel in cortical slices from P22 - 23 WT ( n = 28 ; N = 11 ) KI mice ( n = 27 ; N = 9 ) . STC τdecay is 21 % higher in KI compared to WT astrocytes ( unpaired t - : P < 0 . 0001 ) .

O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O

(C) τdecay of the STC isolated using an exponential waveform approximating the average TBOA-insensitive current in WT and KI mice. Top trace, average normalized TBOA-insensitive current obtained by pooling the normalized TBOA-insensitive currents recorded in 16 WT and KI cells. This current was best fitted by an exponentially rising function (1-exp (-t/τrise)) with τrise= 2.35 ms (in green). The STC was obtained by subtracting from the total current elicited in the astrocyte (a: same representative trace as in (B)) the exponential function A(1-exp (-t/τrise)) with τrise = 2.35 ms and A equal to the maximal current measured in the astrocyte at about 60 ms after stimulation (trace c); the decay of the STC (trace a-c) was best fitted by a single exponential function with τdecay = 6.49 ms (in red). The bar plot shows the average values of τdecay of the STC isolated as shown in the top panel in cortical slices from P22-23 WT (n = 28; N=11) and KI mice (n = 27; N=9). STC τdecay is 21% higher in KI compared to WT astrocytes (unpaired t-test: P < 0.0001).


( A ) Isolation of STC elicited by last of a high - frequency train of action potentials . Left : superimposed representative traces of inward current evoked in an astrocyte ( held at - 90 mV ) by extracellular stimulation with a train of 10 pulses ( trace a : black ) a train of 9 pulses ( trace b : brown ) at 50 Hz in a WT cortical slice . inward current elicited by 10th was obtained by subtracting current elicited by 9 pulses from that elicited by 10 pulses ( trace a - b ) . Right : STC elicited by 10th ( trace a - b - c ) was obtained by subtracting exponential function that simulates TBOA - insensitive current elicited by a single ( trace c , obtained as in Figure 1C ) to 10 - 9 pulses difference current ( trace a - b ) . decay of STC elicited by 10th was best fitted by a single exponential function with τdecay = 8 . 04 ms ( in red ) .

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

(A) Isolation of the STC elicited by the last pulse of a high-frequency train of action potentials. Left: superimposed representative traces of the inward current evoked in an astrocyte (held at -90 mV) by extracellular stimulation with a train of 10 pulses (trace a: black) and a train of 9 pulses (trace b: brown) at 50 Hz in a WT cortical slice. The inward current elicited by the 10th pulse was obtained by subtracting the current elicited by 9 pulses from that elicited by 10 pulses (trace a-b). Right: The STC elicited by the 10th pulse (trace a-b-c) was obtained by subtracting the exponential function that simulates the TBOA-insensitive current elicited by a single pulse (trace c, obtained as in Figure 1C) to the 10-9 pulses difference current (trace a-b). The decay of the STC elicited by the 10th pulse was best fitted by a single exponential function with τdecay = 8.04 ms (in red).


( B ) τdecay of STC elicited by 10th of 50 Hz ( τ10 ( 50 Hz ) , left panel ) 100 Hz ( τ10 ( 100 Hz ) , right panel ) trains in layer 1 astrocytes in acute cortical slices from P22 - 23 WT ( n = 23 ; N = 10 for 50 Hz ; n = 18 ; N = 8 for 100 Hz ) KI ( n = 21 ; N = 9 for 50 Hz n = 14 ; N = 7 for 100 Hz ) mice . STC elicited by 10th was obtained as described in ( A ) . STC τ10 ( 50 Hz ) τ10 ( 100 Hz ) are 30 % 37 % higher in KI compared to WT mice , respectively ( unpaired t - : P < 0 . 0001 in both cases ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

(B) τdecay of the STC elicited by the 10th pulse of 50 Hz (τ10 (50 Hz), left panel) and 100 Hz (τ10 (100 Hz), right panel) trains in layer 1 astrocytes in acute cortical slices from P22-23 WT (n = 23; N=10 for 50 Hz; n = 18; N=8 for 100 Hz) and KI (n = 21; N=9 for 50 Hz and n = 14; N=7 for 100 Hz) mice. The STC elicited by the 10th pulse was obtained as described in (A). STC τ10 (50 Hz) and τ10 (100 Hz) are 30% and 37% higher in KI compared to WT mice, respectively (unpaired t-test: P < 0.0001 in both cases).


( A ) Simultaneous visualization of GLT - 1a + ( green ) VGLUT1 + ( red ) in first somatic sensory cortex ( SI ) of a WT a KI mouse ( P35 ) . Arrows point to some GLT - 1a + overlaying with VGLUT1 + ( i . e . , GLT - 1a / VGLUT1 related ) ; framed regions ( enlarged below ) are examples of GLT - 1a / VGLUT1 related ( arrowheads ) . microscopic fields are from layers II / III . Scale bars : 3 . 5 µm for left right panels 1 µm for enlarged framed areas . ( B ) of GLT - 1a + overlaying with VGLUT1 in P35 WT KI mice . Left , of GLT - 1a + overlaying with VGLUT1 is comparable in WT KI mice ( data were obtained from 14 18 fields of 20 µm x 20 µm from 2 WT 2 KI mice ( 4 sections / ) , respectively ) ( unpaired t - : P = 0 . 52 ) . Right , of GLT - 1a / VGLUT1 related is 19 % reduced in KI mice ( 169 GLT - 1a + analyzed from 2 mice ) compared to WT ( 174 GLT - 1a + analyzed from 2 mice ) ( Mann - Withney U : P = 0 . 016 ) .

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

(A) Simultaneous visualization of GLT-1a+ (green) and VGLUT1+ puncta (red) in first somatic sensory cortex (SI) of a WT and a KI mouse (P35). Arrows point to some GLT-1a+ puncta overlaying with VGLUT1+ puncta (i.e., GLT-1a/VGLUT1 related puncta); framed regions (enlarged below) are examples of GLT-1a/VGLUT1 related puncta (arrowheads). All microscopic fields are from layers II/III. Scale bars: 3.5 µm for left and right panels and 1 µm for enlarged framed areas.(B) Percentage and size of GLT-1a+ puncta overlaying with VGLUT1 in P35 WT and KI mice. Left, percentage of GLT-1a+ puncta overlaying with VGLUT1 is comparable in WT and KI mice (data were obtained from 14 and 18 fields of 20 µm x 20 µm from 2 WT and 2 KI mice (4 sections/animal), respectively) (unpaired t-test: P = 0.52). Right, size of GLT-1a/VGLUT1 related puncta is 19 % reduced in KI mice (169 GLT-1a+ puncta analyzed from 2 mice) compared to WT (174 GLT-1a+ puncta analyzed from 2 mice) (Mann-Withney U test: P = 0.016).


( A ) Distribution of GLT - 1agold particles in astrocytic processes contacting asymmetric synapses ( perisynaptic astrocytic processes : PAP ) in SI of 5 WT 5 KI ( 2 , 3 ) mice ( P34 - 35 ) . In cortical PAP of KI mice density ( particles / µm2 ) of total - associated gold particles ( arrowheads ) are reduced compared to WT mice , whereas density of cytoplasmicgold particles ( arrows ) is comparable to that in WT ( cf Table I for numerical values statistics ) .

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

(A) Distribution of GLT-1agold particles in astrocytic processes contacting asymmetric synapses (perisynaptic astrocytic processes: PAP) in SI of 5 WT and 5 KI (2 male, 3 female) mice (P34-35). In cortical PAP of KI mice the density (particles/µm2) of total and membrane-associated gold particles (arrowheads) are reduced compared to WT mice, whereas the density of cytoplasmicgold particles (arrows) is comparable to that in WT (cf Table I for numerical values and statistics).


( B ) Distribution of GLT - 1agold particles in excitatory axon terminals forming asymmetric synaptic contact ( AxT ) in SI of P34 - 35 WT KI mice . In cortical AxT of KI WT mice , GLT - 1a density is comparable ( arrowheads : - associated gold particles ; arrows : cytoplasmicgold particles ) ( cf Table I ) .

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

(B) Distribution of GLT-1agold particles in excitatory axon terminals forming asymmetric synaptic contact (AxT) in SI of P34-35 WT and KI mice. In cortical AxT of KI and WT mice, GLT-1a density is comparable (arrowheads: membrane-associated gold particles; arrows: cytoplasmicgold particles) (cf Table I).


Left , superimposed representative traces of normalized inward current evoked in a WT ( black trace ) a KI corticalastrocyte ( blue trace ) by extracellular stimulation with a train of 10 pulses at 50 Hz in cortical slices . inset shows in an expanded time scale portion of traces indicated by dotted . slowly decaying current ( τdecay = 2 . 00 s 3 . 04 s for WT KI trace , respectively ) is largely a [ K + ] e - dependent K + current ( IK ) whose decay kinetics provide a measure of rate of K + clearance by astrocytes ( see text ) . Right , time constant of decay of IK elicited by trains of 10 pulses at 50 Hz in corticalastrocytes from P22 - 23 WT ( n = 21 ; N = 12 ) KI mice ( n = 20 ; N = 8 ) . IK τdecay is 22 % higher in KI compared to WT astrocytes ( unpaired t - : P = 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O

Left, superimposed representative traces of the normalized inward current evoked in a WT (black trace) and a KI corticalastrocyte (blue trace) by extracellular stimulation with a train of 10 pulses at 50 Hz in cortical slices. The inset shows in an expanded time scale the portion of the traces indicated by the dotted line. The slowly decaying current (τdecay = 2.00 s and 3.04 s for the WT and KI trace, respectively) is largely a [K+]e-dependent K+current (IK) whose decay kinetics provide a measure of the rate of K+ clearance by astrocytes (see text). Right, time constant of decay of IK elicited by trains of 10 pulses at 50 Hz in corticalastrocytes from P22-23 WT (n = 21; N=12) and KI mice (n = 20; N=8). IK τdecay is 22% higher in KI compared to WT astrocytes (unpaired t-test: P = 0.001).


( A ) of a cortical slice before ( t = 0 ) after ( t = 15 . 6 s ) pressure ejection of a high KCl that elicits a CSD ( top panels ) , showing that propagating CSD is associated with a change in transmittance . Scale bar : 500 µm . traces below show representative changes in intrinsic optical signal ( IOS ) relative to background measured in a WT a KI cortical slice during CSD propagation at increasing times distances from KCl puff , as indicated in color code in right image on top . velocity of CSD propagation , obtained from rate of horizontal spread of change in IOS , in these two representative WT KI slices is 3 . 11 4 . 14 mm / min , respectively . ( B ) Stimulation threshold for CSD induction ( CSD threshold ) rate of CSD propagation ( CSD velocity ) in WT ( n = 24 ; N = 3 ) KI ( n = 27 ; N = 8 ) cortical slices from P22 - 23 mice . CSD threshold is expressed as duration of first KCl eliciting a CSD . CSD threshold is 28 % lower ( Mann Whitney U , P < 0 . 0001 ) CSD velocity 21 % higher ( unpaired t - : P < 0 . 0001 ) in KI compared to WT mice .

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

(A) Images of a cortical slice before (t = 0) and after (t = 15.6 s) pressure ejection of a high KCl pulse that elicits a CSD (top panels), showing that the propagating CSD is associated with a change in light transmittance. Scale bar: 500 µm. The traces below show representative changes in intrinsic optical signal (IOS) relative to background measured in a WT and a KI cortical slice during CSD propagation at increasing times and distances from the KCl puff, as indicated in color code in the right image on the top. The velocity of CSD propagation, obtained from the rate of horizontal spread of the change in IOS, in these two representative WT and KI slices is 3.11 and 4.14 mm/min, respectively.(B) Stimulation threshold for CSD induction (CSD threshold) and rate of CSD propagation (CSD velocity) in WT (n = 24; N=3) and KI (n = 27; N=8) cortical slices from P22-23 mice. CSD threshold is expressed as duration of the first KCl pulse eliciting a CSD. CSD threshold is 28% lower (Mann Whitney U test, P < 0.0001) and CSD velocity 21% higher (unpaired t-test: P< 0.0001) in KI compared to WT mice.


( A ) Western blottings of GLT - 1a in cortical crude synaptic membranes of P39 WT mice following Cef treatment for 8 days . GLT - 1a are significantly increased in mice treated with Cef ( Cef WT , N = 4 ) compared to control saline - injected mice ( Ctr WT , N = 4 ) ( Mann - Withney U : P = 0 . 028 ) .

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

(A) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 WT mice following Cef treatment for 8 days. GLT-1a levels are significantly increased in mice treated with Cef (Cef WT, N = 4) compared to control saline-injected mice (Ctr WT, N = 4) (Mann-Withney U test: P = 0.028).


( B ) Visualization of GLT - 1a + ( green ) VGLUT1 + ( red ) in sections from SI of P45 - 46 WT mice that were treated with saline ( Ctr WT ) or Cef ( Cef WT ) . Arrows point to some GLT - 1a / VGLUT1 related . Framed regions ( enlarged below ) are examples of GLT - 1a / VGLUT1 related ( arrowheads ) . Right , Cef treatment increased significantly of GLT - 1a + overlaying VGLUT1 ( 142 186 GLT - 1a + analyzed from 4 Ctr WT 4 Cef WT mice , respectively ; 3 sections / ) ( Mann - Withney U : P < 0 . 0001 ) . microscopic fields are from layers II / III . Scale bars : 3 . 5 µm for left right panels 1 µm for enlarged framed areas .

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

(B) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in sections from SI of P45-46 WT mice that were treated with saline (Ctr WT) or Cef (Cef WT). Arrows point to some GLT-1a/VGLUT1 related puncta. Framed regions (enlarged below) are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment increased significantly the size of GLT-1a+ puncta overlaying VGLUT1 (142 and 186 GLT-1a+ puncta analyzed from 4 Ctr WT and 4 Cef WT mice, respectively; 3 sections/animal) (Mann-Withney U test: P < 0.0001). All microscopic fields are from layers II/III. Scale bars: 3.5 µm for left and right panels and 1 µm for enlarged framed areas.


( C ) CSD threshold CSD velocity in cortical slices from P30 - 33 KI mice that were injected with saline ( Ctr KI , n = 38 ; N = 7 ) or Cef ( Cef KI , n = 31 ; N = 6 ) . CSD threshold is 12 % higher ( Mann - Withney U : P = 0 . 02 ) in Cef - treated compared to saline - treated KI mice . CSD velocity is not altered by Cef treatment ( P = 0 . 90 ) .

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

(C) CSD threshold and CSD velocity in cortical slices from P30-33 KI mice that were injected with saline (Ctr KI, n = 38; N=7) or Cef (Cef KI, n = 31; N=6). CSD threshold is 12% higher (Mann-Withney U test: P = 0.02) in Cef-treated compared to saline-treated KI mice. CSD velocity is not altered by Cef treatment (P = 0.90).


( A ) Distribution of GLT - 1agold particles in PAP of saline - injected ( Ctr KI , N = 4 ) Cef - treated KI mice ( Cef KI , N = 4 ) ( P45 - 46 ) . Cef treatment does not modify density of total , ( arrows ) - associated ( arrowheads ) gold particles in PAP of KI mice ( cf Table II ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O B-ORGANISM O O O O O O

(A) Distribution of GLT-1agold particles in PAP of saline-injected (Ctr KI, N=4) and Cef-treated KI mice (Cef KI, N=4) (P45-46). Cef treatment does not modify the density of total, cytoplasmic (arrows) and membrane-associated (arrowheads) gold particles in PAP of KI mice (cf Table II).


( B ) Distribution of GLT - 1agold particles in AxT in SI of Ctr KI Cef KI KI mice . density of - associated gold particles ( arrowheads ) is increased in Cef - treated KI mice ( cf Table II ) .

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

(B) Distribution of GLT-1agold particles in AxT in SI of Ctr KI and Cef KI KI mice. The density of the membrane-associated gold particles (arrowheads) is increased in Cef-treated KI mice (cf Table II).


( C ) Western blottings of GLT - 1a in cortical crude synaptic membranes of P39 KI mice following Cef treatment . GLT - 1a are similar in Cef - treated ( Cef KI , N = 4 ) saline - injected ( Ctr KI , N = 4 ) ( Mann - Withney U : P = 0 . 83 ) .

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

(C) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 KI mice following Cef treatment. GLT-1a levels are similar in Cef-treated (Cef KI, N=4) and and saline-injected (Ctr KI, N=4) (Mann-Withney U test: P = 0.83).


( D ) Visualization of GLT - 1a + ( green ) VGLUT1 + ( red ) in KI mice that received saline ( Ctr KI ) in Cef - treated KI mice ( Cef KI ) ( P45 - 46 ) . Arrows point to some GLT - 1a / VGLUT1 related ; framed regions are examples of GLT - 1a / VGLUT1 related ( arrowheads ) . Right , Cef treatment does not increase of GLT - 1a + overlaying with VGLUT1 ( 160 175 GLT - 1a + analyzed from same 4 Ctr KI 4 Cef KI used for post - embedding electron microscopy ; 3 sections / ) ( Mann - Withney U : P = 0 . 31 ) . Scale bar : 3 . 5 µm for left right panels 1 µm for enlarged framed areas . microscopic fields are from layers II / III .

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

(D) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in KI mice that received saline (Ctr KI) and in Cef-treated KI mice (Cef KI) (P45-46). Arrows point to some GLT-1a/VGLUT1 related puncta; framed regions are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment does not increase the size of GLT-1a+ puncta overlaying with VGLUT1 (160 and 175 GLT-1a+ puncta analyzed from the same 4 Ctr KI and 4 Cef KI used for post-embedding electron microscopy analysis; 3 sections/animal) (Mann-Withney U test: P = 0.31). Scale bar: 3.5 µm for left and right panels and 1 µm for enlarged framed areas. All microscopic fields are from layers II/III.


( A ) τdecay of STC evoked by single stimulation in layer 1 astrocytes in acute cortical slices from P22 - 23 WT mice before ( Ctr WT ) after application of 2 . 5 µM DL - TBOA ( TBOA 2 . 5 ) ( n = 4 ; N = 2 ) ( right panel ) . traces on left are corresponding average normalized STCs , isolated as in Figure 1C . STC τdecay in TBOA 2 . 5 is 32 ± 2 % higher than in Ctr WT ( paired t - : P = 0 . 002 ) . left top panel shows time course of τdecay of transient component ( due to STC ) of current recorded in an astrocyte during a representative experiment in which TBOA 2 . 5 was applied at time indicated by horizontal bar . steady - state effect was reached within 10 - 15 minutes from beginning of drug perfusion .

O O O O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22-23 WT mice before (Ctr WT) and after application of 2.5 µM DL-TBOA (TBOA 2.5) (n = 4; N=2) (right panel). The traces on the left are the corresponding average normalized STCs, isolated as in Figure 1C. The STC τdecay in TBOA 2.5 is 32 ± 2 % higher than in Ctr WT (paired t-test: P = 0.002). The left top panel shows the time course of τdecay of the transient component (due to the STC) of the current recorded in an astrocyte during a representative experiment in which TBOA 2.5 was applied at the time indicated by the horizontal bar. The steady-state effect was reached within 10-15 minutes from the beginning of the drug perfusion.


( B ) CSD threshold velocity measured in cortical slices from P22 - 23 WT mice after perfusion for 20 minutes with extracellular solution without ( Ctr WT : n = 23 ; N = 15 ) with 2 . 5 µM DL - TBOA ( TBOA 2 . 5 : n = 25 ; N = 8 ) . CSD threshold in TBOA 2 . 5 is 36 % lower than in Ctr WT ( Mann - Withney U : P < 0 . 0001 ) CSD velocity is 20 % higher ( unpaired t - : P < 0 . 0001 ) .

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

(B) CSD threshold and velocity measured in cortical slices from P22-23 WT mice after perfusion for 20 minutes with extracellular solution without (Ctr WT: n = 23; N = 15) and with 2.5 µM DL-TBOA (TBOA 2.5: n = 25; N = 8). CSD threshold in TBOA 2.5 is 36 % lower than in Ctr WT (Mann-Withney U test test: P < 0.0001) and CSD velocity is 20% higher (unpaired t-test: P < 0.0001).


( C ) τdecay of STC evoked by single stimulation in layer 1 astrocytes in acute cortical slices from P22 - 23 WT mice before ( Ctr WT ) after application of 1 . 5 µM DL - TBOA ( TBOA 1 . 5 ) ( n = 4 ; N = 4 ) . STC τdecay in TBOA 1 . 5 is 22 ± 3 % higher than in Ctr WT ( paired t - : P = 0 . 004 ) .

O O O O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

(C) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22-23 WT mice before (Ctr WT) and after application of 1.5 µM DL-TBOA (TBOA 1.5) (n = 4; N=4). The STC τdecay in TBOA 1.5 is 22 ± 3 % higher than in Ctr WT (paired t-test: P = 0.004).


( D ) CSD threshold velocity measured in cortical slices from P22 - 23 WT mice after perfusion for 20 minutes with extracellular solution without ( Ctr WT : n = 23 ; N = 15 ) with 1 . 5 µM DL - TBOA ( TBOA 1 . 5 : n = 18 ; N = 6 ) . CSD threshold in TBOA 1 . 5 is 23 % lower than in Ctr WT ( Mann - Withney U : P < 0 . 0001 ) CSD velocity is 13 % higher ( unpaired t - : P = 0 . 003 ) .

(D) CSD threshold and velocity measured in cortical slices from P22-23 WT mice after perfusion for 20 minutes with extracellular solution without (Ctr WT: n = 23; N = 15) and with 1.5 µM DL-TBOA (TBOA 1.5: n = 18; N = 6). CSD threshold in TBOA 1.5 is 23 % lower than in Ctr WT (Mann-Withney U test test: P < 0.0001) and CSD velocity is 13 % higher (unpaired t-test: P = 0.003).


( A Total PD - L1 , PARP from colon ( Colo - 205 ) , lung ( A549 ) , pancreatic ( PANK1 ) lysates treated with indicated DR5 agonist antibodies named Lexa ( lexatumumab ) , KMTR2 GAPDH is loading

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(A Total PD-L1, PARP from colon (Colo-205), lung (A549), pancreatic (PANK1) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2 GAPDH is loading control


Total PD - L1 , PARP from triple negative breast cancer ( MDA - MB - 436 ) lysates treated with indicated DR5 agonist antibodies named Lexa ( lexatumumab ) , KMTR2 , BaCa , GAPDH is loading

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Total PD-L1, PARP from triple negative breast cancer cell (MDA-MB-436) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2, BaCa, GAPDH is loading control


C ) Total PD - L1 , PARP , caspase - 3 from ovarian ( Cavo - 3 ) lysates treated with indicated DR5 agonist antibodies named tigatuzumab . GAPDH is loading

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C) Total PD-L1, PARP, cleaved caspase-3 from ovarian cell (Cavo-3) lysates treated with indicated DR5 agonist antibodies named tigatuzumab. GAPDH is loading control


( D ) Total PD - L1 , CD47 , Calreticulin , PARP from MDA - MB - 436 lysates treated with indicated DR5 agonist antibodies ± caspase inhibitor Z - VAD . GAPDH is loading control .

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(D) Total PD-L1, CD47, Calreticulin, PARP from MDA-MB-436 cell lysates treated with indicated DR5 agonist antibodies ± caspase inhibitor Z-VAD. GAPDH is loading control.


( E ) Total PD - L1 western blotting signal from lysates of OVCAR - 3 ( n = 3 ) MDA - MB - 436 ( n = 3 ) after 6 hrs treatment of 100nM lexa ( DR5 agonist antibody ) was normalized to GAPDH .

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(E) Total PD-L1 western blotting signal from the lysates of OVCAR-3 (n=3) and MDA-MB-436 (n=3) cells after 6 hrs treatment of 100nM lexa (DR5 agonist antibody) was normalized to GAPDH.


( F ) Surface biotinylation of PD - L1 from indicated after indicated DR5 agonist treatments . caspase - 3 PARP indicates activation of DR5 signaling . In lane 3 4 , KMTR2 was pre - neutralized either with recombinant DR5 ( rDR5 ) or recombinant FOLR1 ( rFOLR1 ) .

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(F) Surface biotinylation of PD-L1 from indicated tumor cells after indicated DR5 agonist treatments. Cleaved caspase-3 and PARP indicates activation of DR5 signaling. In lane 3 and 4, KMTR2 was pre-neutralized either with recombinant DR5 (rDR5) or recombinant FOLR1 (rFOLR1).


( G ) Representative flow cytometry plots of PD - L1 from two different lines treated with indicated DR5 antibodies . Secondary alone IgG1 control are included .

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(G) Representative flow cytometry plots of PD-L1 from two different tumor cell lines treated with indicated DR5 antibodies. Secondary alone and IgG1 control are included.


( H ) Relative surface PD - L1 % , after DR5 agonist treatments Relative signal is normalized to surface PD - L1 in IgG1 treated in corresponding horizontal in samples indicated mean ( n = 3 - 4 ) .

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(H) Relative surface PD-L1 % cells, after DR5 agonist treatments Relative signal is normalized to surface PD-L1 in IgG1 treated cells in corresponding tumor cells and horizontal line in samples indicated mean (n=3-4).


( J ) Relative surface PD - L1 % in indicated tumors after DR5 agonist treatments Relative signal is normalized to surface PD - L1 in IgG1 treated tumors horizontal in samples indicated mean ( n = 2 - 6 )

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(J) Relative surface PD-L1 % cells in indicated tumors after DR5 agonist treatments Relative signal is normalized to surface PD-L1 in IgG1 treated tumors and horizontal line in samples indicated mean (n=2-6)


( K ) ER ( - ) , PR ( - ) HER2 ( - ) UCD52 derived tissue was xenografted in breast fat pad of NOD . Cg - Prkdcscid Il2rgtm1Wjl / SzJ mice , following by treatment with IgG1 or KMTR2 ( 50μg , 4 doses ) . Harvested tumors were analyzed for PD - L1 , CD47 PARP in lysates .

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(K) ER (-), PR (-) and HER2 (-) UCD52 patient derived tumor tissue was xenografted in breast fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, following by treatment with IgG1 or KMTR2 (50μg, 4 doses). Harvested tumors were analyzed for PD-L1, CD47 and PARP in lysates.


( L ) KMTR2 treated ( 50μg , 4 doses ) UCD52 TNBC PDX tumors were stained for PD - L1 using immunohistochemistry ( ) .

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(L) KMTR2 treated (50μg, 4 doses) UCD52 TNBC PDX tumors were stained for PD-L1 using immunohistochemistry (IHC).


( M ) Total PD - L1 CD47 blotting from DR5 resistant MDA - MB - 436 lines after indicated DR5 agonist treatment ( 50nM , 6hr ) .

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(M) Total PD-L1 and CD47 blotting analysis from DR5 resistant MDA-MB-436 cell lines after indicated DR5 agonist treatment (50nM, 6hr).


( C ) Luciferase activity of reporter lines from - Jurkat co - culture using MDA - MB - 436 OVCAR3 after treatment ( 50nM ) with indicated DR5 agonists ( tiga : tigatuzumab , AMG : AMG655 , KMTR1 , Lexa : Lexatumumab ) . background luciferase signal from untreated ( due to basal surface PD - L1 ) was subtracted . Various controls treatments ( IgG1 , anti - PD - L1 , anti - EGFR ) are also shown ( n = 3 ) . ( D ) Same as ( C ) except were pre - treated with indicated inhibitors for AKT , ERK , mTOR , MEK , STAT3 , PARP - i NF - Kβ along with DR5 agonist KMTR2 antibody prior to co - culture .

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

(C) Luciferase activity of reporter lines from tumor cell-Jurkat cell co-culture assay using MDA-MB-436 and OVCAR3 cells after treatment (50nM) with indicated DR5 agonists (tiga: tigatuzumab, AMG: AMG655, KMTR1, Lexa: Lexatumumab). The background luciferase signal from untreated cells (due to basal surface PD-L1) was subtracted. Various controls treatments (IgG1, anti-PD-L1, anti-EGFR) are also shown (n=3). (D) Same as (C) except tumor cells were pre-treated with indicated inhibitors for AKT, ERK, mTOR, MEK, STAT3, PARP-i and NF-Kβ along with DR5 agonist KMTR2 antibody prior to co-culture.


MDA - MB - 436 were treated with TNFα indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 , CSN5 GAPDH is loading control in

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MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1, CSN5 GAPDH is loading control in E


MDA - MB - 436 were treated with TNFα indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 phosphorylated p65 , total p65 , caspase - 3 PARP . Tubulin is loading control in F .

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MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1 phosphorylated p65, total p65, cleaved caspase-3 and PARP. Tubulin is loading control in F.


MDA - MB - 436 were treated with TNFα indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 , CSN5 , phosphorylated p65 PARP . GAPDH is loading control in G .

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MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1, CSN5, phosphorylated p65 and PARP. GAPDH is loading control in G.


( H ) Flow cytometry of MDA - MB - 436 treated with TNFα ± MG132 ( top ) indicated DR5 agonist ± MG132 ( bottom ) .

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(H) Flow cytometry analysis of MDA-MB-436 cells treated with TNFα ± MG132 (top) and indicated DR5 agonist ± MG132 (bottom).


( I ) Total PD - L1 from OVCAR3 lysates was analyzed after treatment of indicated DR5 agonist ± MG132 .

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(I) Total PD-L1 from OVCAR3 cell lysates was analyzed after treatment of indicated DR5 agonist ± MG132.


( J ) Flow cytometry surface PD - L1 of OVCAR - 3 treated with indicated DR5 agonist ± MG132 .

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

(J) Flow cytometry surface PD-L1 analysis of OVCAR-3 cells treated with indicated DR5 agonist ± MG132.


( K ) MDA - MB - 436 were treated with MG132 for indicated times total PD - L1 from lysates was analyzed . GAPDH is loading control .

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(K) MDA-MB-436 cells were treated with MG132 for indicated times and total PD-L1 from lysates was analyzed. GAPDH is loading control.


( L ) Immunoblotting of DR5 confirming generation of knock out ( DR5 - KO ) lines .

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(L) Immunoblotting of DR5 confirming generation of knock out (DR5-KO) cell lines.


( M ) viability of DR5 - KO DR5 - WT ( n = 3 ) .

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(M) Cell viability analysis of DR5-KO and DR5-WT cells (n=3).


( N ) DR5 - KO WT were treated with TNFα indicated DR5 agonist followed by flow cytometry using PD - L1 specific antibodies .

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(N) DR5-KO and WT cells were treated with TNFα and indicated DR5 agonist followed by flow cytometry using PD-L1 specific antibodies.


( O ) TNBC WT DR5 - KO ( MDA - MB - 436 ) were treated with either DR5 agonist or TNFα as indicated . Lysates were analyzed for PD - L1 , S5a DR5 . GAPDH is loading control .

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(O) TNBC WT and DR5-KO (MDA-MB-436) cells were treated with either DR5 agonist or TNFα as indicated. Lysates were analyzed for PD-L1, S5a and DR5. GAPDH is loading control.


( P ) OVCAR - 3 MDA - MB - 436 ( WT DR5 - KO ) were treated with KMTR2 followed by ubiquitin S5a immunoblotting from total lysates . GAPDH is loading control .

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

(P) OVCAR-3 and MDA-MB-436 (WT and DR5-KO) cells were treated with KMTR2 followed by ubiquitin and S5a immunoblotting from total lysates. GAPDH is loading control.


ApoEVs isolated after IgG1 KMTR2 treatment ( OVCAR - 3 ) were blotted against CD63 PD - L1 in dot blots .

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ApoEVs isolated after IgG1 and KMTR2 treatment (OVCAR-3) were blotted against CD63 and PD-L1 in dot blots.


ApoEVs isolated after IgG1 tigatuzumab treatment ( MDA - MB - 436 ) were run on western blotted against CD63 PD - L1 .

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ApoEVs isolated after IgG1 and tigatuzumab treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.


ApoEVs isolated after IgG1 lexatumumab ( D ) treatment ( MDA - MB - 436 ) were run on western blotted against CD63 PD - L1 .

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

ApoEVs isolated after IgG1 and lexatumumab (D) treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.


( F ) ApoEVs isolated from DR5 sensitive grown in Met - HPG + media were added on to DR5 - KO ( growing in regular media ) . After 48 hrs flow cytometry was carried out with HPG catalyzing dye . HPG incorporation from flow cytometry data confirms ApoEVs transfer from growing in Met - HPG + ( DR5 - WT ) to DR5 - KO .

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(F) ApoEVs isolated from DR5 sensitive tumor cells grown in Met-HPG+ media were added on to DR5-KO cells (growing in regular media). After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye. HPG incorporation from flow cytometry data confirms ApoEVs transfer from cells growing in Met-HPG+ (DR5-WT) to DR5-KO cells.


( G ) ApoEVs isolated from DR5 - WT OVCAR - 3 ( KMTR2 treated ) were added on to DR5 - KO ( MDA - MB - 231 ) for indicated times ( 24 - 96 hrs ) to analyze PD - L1 transfer kinetics from

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

(G) ApoEVs isolated from DR5-WT OVCAR-3 cells (KMTR2 treated) were added on to DR5-KO (MDA-MB-231) cells for indicated times (24-96 hrs) to analyze PD-L1 transfer kinetics from ApoEVs


after addition of MDA - MB - 436 derived ApoEVs ( treated with combination of KMTR2 + lexatumumab DR5 agonists or IgG1 ) on to DR5 - KO MDA - MB - 231 , total lysates were immunoblotted for PD - L1 after 24 hrs . KMTR2 + lexatumumab ( n = 3 ) , IgG1 ( n = 2 ) .

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

after addition of MDA-MB-436 derived ApoEVs (treated with combination of KMTR2+lexatumumab DR5 agonists or IgG1) on to DR5-KO MDA-MB-231 cells, total lysates were immunoblotted for PD-L1 after 24 hrs. KMTR2+lexatumumab (n=3), IgG1 (n=2).


( I ) Left : PD - L1 surface histogram shows direct antibody treatment on DR5 - KO . Right : PD - L1 surface histogram from DR5 - KO after treatment ( 24 hrs ) with ApoEVs isolated from DR5 sensitive after either Lexa or KMTR2 treatment .

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

(I) Left: PD-L1 surface histogram shows direct antibody treatment on DR5-KO cells. Right: PD-L1 surface histogram from DR5-KO cells after treatment (24 hrs) with ApoEVs isolated from DR5 sensitive cells after either Lexa or KMTR2 treatment.


ApoEVs treated were analyzed in PD - 1 reporter co - culture assays for luciferase

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ApoEVs treated tumor cells were analyzed in PD-1 reporter co-culture assays for luciferase signal


( K ) MDA - MB - 436 were treated with KMTR2 for 6 hrs along with ZDEVD for indicated times . - ZDEVD shows final time of KMTR2 exposure to in absence inhibitor . Lysates were immunoblotted for caspase - 3 , 8 , PARP , PD - L1 , S5a , ROCK1 GAPDH ( n = 3 ) . u . c : uncleaved , c : , n . s : non - specific

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(K) MDA-MB-436 cells were treated with KMTR2 for 6 hrs and along with ZDEVD for indicated times. - ZDEVD shows final time of KMTR2 exposure to cells in absence inhibitor. Lysates were immunoblotted for cleaved caspase-3, 8, PARP, PD-L1, S5a, ROCK1 and GAPDH (n=3). u.c: uncleaved, c: cleaved, n.s: non-specific band


( L ) Relative caspase - 8 caspase - 3 activity assays caspase - 8 maintained steady activity , while caspase - 3 activity required at least 40 ( + ) mins of DR5 agonist treatment without ZDEVD ( n = 3 ) .

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

(L) Relative caspase-8 and caspase-3 activity assays caspase-8 maintained steady activity, while caspase-3 activity required at least 40 (+) mins of DR5 agonist treatment without ZDEVD (n=3).


( M ) After indicated treatments with KMTR2 ( DR5 agonist ) ZDEVD ( caspase - 3 preferred inhibitor ) , were allowed to grow 24 hrs , followed by viability .

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(M) After indicated treatments with KMTR2 (DR5 agonist) and ZDEVD (caspase-3 preferred inhibitor) , cells were allowed to grow 24 hrs, followed by cell viability analysis.


( A KMTR2 , KMTR2 + Z - VAD treated MDA - MB - 436 lysates for indicated early time points were analyzed for caspase - 8 , caspase - 3 , ROCK1 PARP as indicated .

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(A KMTR2, KMTR2+Z-VAD treated MDA-MB-436 lysates for indicated early time points were analyzed for caspase-8, caspase-3, ROCK1 PARP as indicated.


B ) lexatumumab treated OVCAR3 lysates for indicated early time points were analyzed for caspase - 3 , ROCK1 , pMLC , PARP , PD - L1 CMTM6 etc . as indicated .

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B) lexatumumab treated OVCAR3 lysates for indicated early time points were analyzed for caspase-3, ROCK1, pMLC, PARP, PD-L1 and CMTM6 etc. as indicated.


( C ) OVCAR3 were treated with pMLC activating ionophore A23187 ( positive control ) lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1 . Lysates were later analyzed for ROCK1 , pMLC , caspase - 3 PARP with GAPDH as loading control . Blue arrows indicate activated ROCK1 caspase - 3 .

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(C) OVCAR3 cells were treated with pMLC activating ionophore A23187 (positive control) and lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1. Lysates were later analyzed for ROCK1, pMLC, caspase-3 and PARP with GAPDH as loading control. Blue arrows indicate cleaved and activated ROCK1 and caspase-3.


( D ) MDA - MB - 436 OVCAR3 were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment ( 20nM ) . After 6 hours flow cytometry was used to analyze surface PD - L1 . ( GSK269 : GSK269962A , GSK429 : GSK429286A )

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(D) MDA-MB-436 and OVCAR3 cells were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment (20nM). After 6 hours flow cytometry was used to analyze surface PD-L1. (GSK269: GSK269962A, GSK429: GSK429286A)


( E ) MDA - MB - 436 survival after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors , error bars indicate SD ( n = 2 ) . ( GSK269 : GSK269962A , GSK429 : GSK429286A )

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(E) MDA-MB-436 cell survival assay after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors, error bars indicate SD (n=2). (GSK269: GSK269962A, GSK429: GSK429286A)


Cultured MDA - MB - 436 were treated with KMTR2 ( IgG1 - Fc ) or IgG1 control ± ROCK1i ( GSK269962 ) for 6 hrs . After 6 hrs , 500nM anti - PD - L1 avelumab ( IgG4 - Fc ) was added to media for additional 1 hr . lysates were pulled down with anti - hu - IgG4 specific beads . ( G ) Immunoprecipitated lysates leftover supernatant after various treatments were run at same time followed by blotting with ROCK1 , PD - L1 CMTM6 . Total lysates as a control were also generated in exactly similar conditions run next to supernatant IP samples . Blue arrows indicate activated ROCK1 .

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

Cultured MDA-MB-436 cells were treated with KMTR2 (IgG1-Fc) or IgG1 control ± ROCK1i (GSK269962) for 6 hrs. After 6 hrs, 500nM anti-PD-L1 avelumab (IgG4-Fc) was added to the media for additional 1 hr. Cellular lysates were pulled down with anti-hu-IgG4 specific beads. (G) Immunoprecipitated lysates and leftover supernatant after various treatments were run at the same time followed by blotting with ROCK1, PD-L1 and CMTM6. Total lysates as a control were also generated in exactly similar conditions and run next to supernatant and IP samples. Blue arrows indicate cleaved and activated ROCK1.


( H ) Immunoprecipitation assays with anti - PD - L1 ( avelumab ) using OVCAR - 3 lines . Blue arrow indicates activated ROCK1 .

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(H) Immunoprecipitation assays with anti-PD-L1 (avelumab) using OVCAR-3 cell lines. Blue arrow indicates cleaved and activated ROCK1.


( J ) Relative luciferase activity ata is from 4 different DR5 agonist antibodies used in combination of two different ROCK1i inhibitors ( n = 3 ) .

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

(J) Relative luciferase activity ata is from 4 different DR5 agonist antibodies used in combination of two different ROCK1i inhibitors (n=3).


( K ) regression efficacy of MD5 - 1 ( 50μg ) , GSK269962A ( < 2mg / kg ) MD5 - 1 + GSK269962A ( 50μg + 2mg / kg ) 4T1 tumors ( n = 4 - 7 ) . 6 - 8 old BALB / c mice bearing 4T1 tumors ( ~ 100mm3 ) were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . GSK269962A ( ROCK1i ) was injected directly into tumors . volumes were quantified at indicated days by caliper measurements .

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

(K) Tumor regression efficacy of MD5-1 (50μg), GSK269962A (<2mg/kg) and MD5-1+ GSK269962A (50μg + 2mg/kg) 4T1 tumors (n=4-7). 6-8 weeks old BALB/c mice bearing 4T1 tumors (~100mm3) were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). GSK269962A (ROCK1i) was injected directly into the tumors. Tumor volumes were quantified at indicated days by caliper measurements.


( B - D ) FACs plots confirming expression of human DR5 ( huDR5 ) Chi - G4S - DR5 in mouse 4T1 . Chi - DR5 was not expressed on surface ( C ) .

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(B-D) FACs plots confirming expression of human DR5 (huDR5) and Chi-G4S-DR5 in mouse 4T1 cells. Chi-DR5 was not expressed on cell surface (C).


( E ) viability of huDR5 Chi - G4S - DR5 stable 4T1 with indicated human DR5 agonists lexatumumab KMTR2 ( n = 3 ) .

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(E) Cell viability analysis of huDR5 and Chi-G4S-DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 (n=3).


( F ) Comparison of growth of grafted huDR5 Chi - G4S - DR5 stable 4T1 in Balb / c mice ( n = 3 ) .

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(F) Comparison of tumor growth of grafted huDR5 and Chi-G4S-DR5 stable 4T1 cells in Balb/c mice (n=3).


( G ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 150 - 200mm3 ) were treated with 4 doses ( 100μg ) of IgG1 , lexatumumab , lexatumumab + avelumab followed by recovery surface PD - L1 using flow

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(G) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~150-200mm3) were treated with 4 doses (100μg) of IgG1, lexatumumab, lexatumumab +avelumab followed by tumor recovery and surface PD-L1 analysis using flow cytometry