Immunofluorescence against CB1 following markers : Calbindin for Purkinje ( E ) , GFAP for astrocytes ( F ) F4 / 80 for microglia ( G ) in cerebellum of WT ASM - KO mice . DAPI stains nuclei . Graphs to left show mean ± associated to CB1 in Purkinje ( shown by arrows , E ) , astrocytes ( F ) microglia ( G ) expressed as of values obtained in WT mice . Graphs to right show Mander ' s coefficient that indicates degree of co - localization between CB1 markers for Purkinje ( E ) , astrocytes ( F ) microglia ( G ) ( E : * * PFluorecence = 0 . 0082 , * PMander ' s Coefficient = 0 . 0158 ; F : * PMander ' s Coefficient = 0 . 0115 ) ; n = 5 mice per group , Student ' s t - ) . Scale bar , 100 μm .

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

Immunofluorescence images against CB1 and the following cellular markers: Calbindin for Purkinje cells (E), GFAP for astrocytes (F) and F4/80 for microglia (G) in the cerebellum of WT and ASM-KO mice. DAPI stains cell nuclei. Graphs to the left show mean ± SEM intensity associated to CB1 in the Purkinje cells (shown by white arrows, E), astrocytes (F) and microglia (G) expressed as percentage of the values obtained in WT mice. Graphs to the right show the Mander's coefficient that indicates degree of co-localization between CB1 and the cellular markers for Purkinje cells (E), astrocytes (F) and microglia (G) (E: **PFluorecence intensity = 0.0082, *PMander's Coefficient = 0.0158; F: *PMander's Coefficient = 0.0115); n = 5 mice per group, Student's t-test). Scale bar, 100 μm.


H . Immunofluorescence against CB1 Purkinje marker calbindin in cerebellum of - matched control ASMD - affected children . Graph shows mean ± associated to CB1 in Purkinje expressed as of values obtained in control child ( 16 15 replicates in control ASMD , respectively ) . Scale bar , 10 μm .

O O B-EXP_ASSAY O B-GENEPROD B-CELL_TYPE O B-GENEPROD O B-TISSUE O O O O B-DISEASE O O B-ORGANISM O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O

H. Immunofluorescence images against CB1 and the Purkinje cell marker calbindin in the cerebellum of age-matched control and ASMD-affected children. Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the values obtained in the control child (16 and 15 replicates in control and ASMD, respectively). Scale bar, 10 μm.


Mean ± CB1 mRNA in cultured hippocampal neurons from WT ASM - KO mice ( * * * * P < 0 . 0001 , n = 5 independent cultures , Student ' s t - ) .

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

Mean ± SEM CB1 mRNA levels in cultured hippocampal neurons from WT and ASM-KO mice (****P < 0.0001, n = 5 independent cultures, Student's t-test).


Western blot against CB1 GAPDH ( used as loading control ) . belong to same blot but to non - consecutive lanes as indicated by boxes . Graph showing mean ± CB1 in cultured neurons from WT ASM - KO mice ( * * * P = 0 . 0009 , n = 5 independent cultures , Student ' s t - ) .

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

Western blot against CB1 and GAPDH (used as loading control). Images belong to the same blot but to non-consecutive lanes as indicated by the boxes. Graph showing mean ± SEM CB1 protein levels in cultured neurons from WT and ASM-KO mice (***P= 0.0009, n = 5 independent cultures, Student's t-test).


Immunofluorescence against CB1 lysosomal marker Lamp1 in cultured neurons from WT ASM - KO mice . TOPRO stains nuclei . Graphs show mean ± associated to CB1 in neuronal processes expressed in arbitrary units ( left ) or Mander ´ s coefficient for colocalization of CB1 with Lamp1 ( right ) ( * * * * P < 0 . 0001 , n = 3 independent cultures , > 30 neurons per culture , Student ' s t - ) . Scale bar , 10 μm .

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

Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from WT and ASM-KO mice. TOPRO stains cell nuclei. Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander´s coefficient for the colocalization of CB1 with Lamp1 (right) (****P < 0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.


Immunofluorescence against CB1 lysosomal marker Lamp1 in Purkinje of cerebellum from WT ASM - KO mice . Graph shows mean ± Mander ´ s coefficient indicative of degree of colocalization of CB1 Lamp1 ( * P = 0 . 0213 , n = 4 mice per group , Student ' s t - ) . Scale bar , 5 μm .

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

Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in Purkinje cells of the cerebellum from WT and ASM-KO mice. Graph shows mean ± SEM Mander´s coefficient indicative of the degree of colocalization of CB1 and Lamp1 (*P= 0.0213, n = 4 mice per group, Student's t-test). Scale bar, 5 μm.


Graphs show mean ± SM , expressed as of WT values , in extracts containing same amount of of cultured neurons from WT ASM - KO mice ( E )

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

Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT and ASM-KO mice (E)


Graphs show mean ± SM , expressed as of WT values , in extracts containing same amount of of cultured neurons from WT mice incubated or not with 40μΜ SM ( F ) .

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

Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT mice incubated or not with 40μΜ SM (F).


Mean ± CB1 mRNA in cultured neurons from WT mice incubated or not with 40μM SM ( * * P = 0 . 0021 , n = 5 independent cultures , Student ' s t - ) .

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

Mean ± SEM CB1 mRNA levels in cultured neurons from WT mice incubated or not with 40μM SM (**P = 0.0021, n = 5 independent cultures, Student's t-test).


Western blot against CB1 GAPDH ( used as loading control ) graph showing mean ± CB1 in cultured neurons from WT mice incubated or not with 40μΜ SM ( n = 2 independent cultures , Student ' s t - )

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

Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice incubated or not with 40μΜ SM (n = 2 independent cultures, Student's t-test)


Immunofluorescence against CB1 Lamp1 in cultured neurons from WT mice incubated or not with 40 μΜ SM . Graphs show mean ± associated to CB1 in neuronal processes expressed in arbitrary units ( left ) or Mander ´ s coefficient for colocalization of CB1 with Lamp1 ( right ) ( * * * P < 0 . 0001 , n = 3 independent cultures , > 30 neurons per culture , Student ' s t - ) . Scale bar , 10 μm .

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

Immunofluorescence images against CB1 and Lamp1 in cultured neurons from WT mice incubated or not with 40 μΜ SM. Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander´s coefficient for the colocalization of CB1 with Lamp1 (right) (***P <0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.


Western blot against CB1 GAPDH ( used as loading control ) graph showing mean ± CB1 in cultured neurons from WT mice treated with vehicle ( veh ) , with 40μM SM ( SM ) , with 0 , 1μM Bafilomycin ( BafA1 ) or with 40μM SM 0 , 1μM Bafilomyecin ( SM + BafA1 ) . Graph shows mean ± CB1 normalized to GAPDH in arbitrary units ( n = 2 independent cultures , one - way ANOVA ) .

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

Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice treated with vehicle (veh), with 40μM SM (SM), with 0,1μM Bafilomycin (BafA1) or with 40μM SM and 0,1μM Bafilomyecin (SM+BafA1). Graph shows mean ± SEM CB1 levels normalized to GAPDH in arbitrary units (n = 2 independent cultures, one - way ANOVA).


Mean ± SM in cultured neurons from ASM - KO mice incubated or not with exogenous SMase ( * P = 0 . 0157 , n = 3 independent cultures , Student ' s t - ) .

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

Mean ± SEM SM levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (*P = 0.0157, n = 3 independent cultures, Student's t-test).


Mean ± CB1 mRNA in cultured neurons from ASM - KO mice incubated or not with exogenous SMase ( * * P = 0 . 0015 , n = 5 independent cultures , Student ' s t - ) .

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

Mean ± SEM CB1 mRNA levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (**P= 0.0015, n = 5 independent cultures, Student's t-test).


Western blot against CB1 GAPDH ( used as loading control ) . belong to same blot but to non - consecutive lanes as indicated by boxes . Graph showing mean ± CB1 normalized to GAPDH in extracts from cultured neurons from ASM - KO mice incubated or not with exogenous SMase ( n = 5 independent cultures , Student ' s t - ) .

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

Western blot against CB1 and GAPDH (used as loading control). Images belong to the same blot but to non-consecutive lanes as indicated by the boxes. Graph showing mean ± SEM CB1 protein levels normalized to GAPDH in extracts from cultured neurons from ASM-KO mice incubated or not with exogenous SMase (n = 5 independent cultures, Student's t-test).


Immunofluorescence against CB1 lysosomal marker Lamp1 in cultured neurons from ASM - KO mice incubated or not with exogenous SMase . DAPI stains nuclei . Graph shows mean ± Mander ´ s coefficient for colocalization of CB1 with Lamp1 ( * P = 0 . 0189 , n = 3 independent cultures , > 30 neurons per culture , Student ' s t - ) . Scale bar , 10 μm .

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

Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from ASM-KO mice incubated or not with exogenous SMase. DAPI stains cell nuclei. Graph shows mean ± SEM Mander´s coefficient for the colocalization of CB1 with Lamp1 (*P = 0.0189, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.


Mean ± SM , expressed as of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle or with indicated concentrations of AEA ( * * * P < 0 . 0001 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) .

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

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle or with the indicated concentrations of AEA (***P<0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).


Mean ± SM , expressed as of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle , inhibitor of NSM GW , AEA or combination of GW AEA ( * * * P < 0 . 0001 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) .

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

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, the inhibitor of NSM GW, AEA or the combination of GW and AEA (***P <0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).


Western blot against NSM GAPDH ( used as loading control ) graph showing mean ± NSM in extracts from cultured neurons treated with vehicle or with 50μM AEA ( * P = 0 . 0230 , n = 6 independent cultures , Student ' s t - ) .

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

Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in extracts from cultured neurons treated with vehicle or with 50μM AEA (*P = 0.0230, n = 6 independent cultures, Student's t-test).


Mean ± SM , expressed as of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle , or with JNJ , PF or URB in presence or absence of AEA ( * * PJNJ = 0 . 0013 , * PPF = 0 . 0143 , * * * PURB = 0 . 0002 , * * PJNJ + AEA = 0 . 0069 , * * PPF + AEA = 0 . 0077 , * * * PURB + AEA < 0 . 0001 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) .

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

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, or with JNJ, PF or URB in the presence or absence of AEA (**PJNJ = 0.0013, *PPF = 0.0143, ***PURB = 0.0002, **PJNJ+AEA = 0.0069, **PPF+AEA = 0.0077, ***PURB+AEA <0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).


Mean ± SM , expressed as of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle , with PF or with SR141716 + PF ( * PPF = 0 . 0109 , * PSR + PF = 0 . 0343 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) .

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

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, with PF or with SR141716 + PF (*PPF = 0.0109, *PSR+PF = 0.0343, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).


Immunofluorescences against dendritic marker MAP2 of cultured neurons from ASM - KO mice treated with vehicle , AEA , JNJ , PF or URB . Scale bar , 50 μm .

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

Immunofluorescences against the dendritic marker MAP2 of cultured neurons from ASM-KO mice treated with vehicle, AEA, JNJ, PF or URB. Scale bar, 50 μm.


Mean ± number of apoptotic measured by TUNEL assays in cultured neurons from ASM - KO mice treated with H2O2 ( as positive control ) , vehicle , AEA , JNJ , PF or URB ( n = 3 independent cultures ) .

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

Mean ± SEM number of apoptotic cells measured by TUNEL assays in cultured neurons from ASM-KO mice treated with H2O2 (as positive control), vehicle, AEA, JNJ, PF or URB (n = 3 independent cultures).


Weekly mean ± of WT ASM - KO mice treated or not with PF ( * * * P < 0 . 0001 , n = 7 mice per group , statistics reflect significant difference between slope of data in PF - treated ASM - KO with respect to other three groups , Linear Regression ) .

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

Weekly mean ± SEM body weight of WT and ASM-KO mice treated or not with PF (***P <0.0001, n = 7 mice per group, statistics reflect the significant difference between the slope of the data in the PF-treated ASM-KO with respect to the other three groups, Linear Regression test).


Mean ± time spent on rod in 4 trials of rotarod by WT ASM - KO mice after two months of vehicle or PF treatment ( * PT3 = 0 . 0488 , * PT4 = 0 . 361 , n = 4 mice per group , two - way ANOVA , Bonferroni post hoc ) .

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

Mean ± SEM time spent on the rod in the 4 trials of the rotarod test by WT and ASM-KO mice after two months of vehicle or PF treatment (*PT3 = 0.0488, *PT4 = 0.361, n = 4 mice per group, two - way ANOVA, Bonferroni post hoc).


Mean ± time spent in new arm of Y maze by WT ASM - KO mice after two months of vehicle or PF treatment ( * * PWTVeh Vs KO Veh = 0 . 0024 , * * PWT PF Vs KO Veh = 0 . 0016 , * PKO PF Vs KO Veh = 0 . 0391 , n = 9 mice per group , two - way ANOVA , Bonferroni post hoc ) .

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

Mean ± SEM time spent in the new arm of the Y maze test by WT and ASM-KO mice after two months of vehicle or PF treatment (**PWTVeh Vs KO Veh = 0.0024, **PWT PF Vs KO Veh = 0.0016, *PKO PF Vs KO Veh = 0.0391, n = 9 mice per group, two - way ANOVA, Bonferroni post hoc).


Mean ± time of immobility in Tail suspension of WT ASM - KO mice after two months of vehicle or PF treatment ( * PWTVeh Vs KO Veh = 0 . 0198 , * * PWT PF Vs KO Veh = 0 . 0067 , n = 6 mice per group , two - way ANOVA , Bonferroni post hoc ) .

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

Mean ± SEM time of immobility in the Tail suspension test of WT and ASM-KO mice after two months of vehicle or PF treatment (*PWTVeh Vs KO Veh = 0.0198, **PWT PF Vs KO Veh = 0.0067, n = 6 mice per group, two - way ANOVA, Bonferroni post hoc).


Mean ± time spent in open arms of Elevated Plus Maze by WT ASM - KO mice after two months of vehicle or PF treatment ( * PWTVeh Vs KO Veh = 0 . 0342 , n = 7 mice per group , two - way ANOVA , Bonferroni post hoc ) .

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

Mean ± SEM time spent in the open arms of the Elevated Plus Maze by WT and ASM-KO mice after two months of vehicle or PF treatment (*PWTVeh Vs KO Veh = 0.0342, n = 7 mice per group, two - way ANOVA, Bonferroni post hoc).


of survival with respect to of in WT ASM - KO mice treated with vehicle or PF ( * * P = 0 . 0031 , n = 5 mice in WT vehicle , WT PF treated KO vehicle , 6 mice in KO PF treated , Mantel - Cox )

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

Percentage of survival with respect to weeks of age in WT and ASM-KO mice treated with vehicle or PF (**P = 0.0031, n = 5 mice in WT vehicle, WT PF treated and KO vehicle, 6 mice in KO PF treated, Mantel-Cox)


Mean ± AEA in cerebellar extracts from WT ASM - KO mice after two months of vehicle or PF treatment ( * * PWT Veh vs KO Veh = 0 . 0063 ; * * * * PWT Veh vs KO PF < 0 . 0001 ; * * * * PWT PF vs KO PF < 0 . 0001 ; * * * * PKO Veh vs KO PF < 0 . 0001 , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) .

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

Mean ± SEM AEA levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (**PWT Veh vs KO Veh = 0.0063; ****PWT Veh vs KO PF < 0.0001; ****PWT PF vs KO PF < 0.0001; ****PKO Veh vs KO PF < 0.0001, n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).


Mean ± SM , expressed as of vehicle values , in cerebellar extracts from WT ASM - KO mice after two months of vehicle or PF treatment ( * * * PWTVeh Vs KO Veh < 0 . 0001 , * PKO Veh Vs KO PF = 0 . 0465 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) .

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

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (***PWTVeh Vs KO Veh <0.0001, *PKO Veh Vs KO PF = 0.0465), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).


Western blot against NSM GAPDH ( used as loading control ) graph showing mean ± NSM in cerebellar extracts from WT ASM - KO mice after two months of vehicle or PF treatment ( n = 3 mice per group , one - way ANOVA , Bonferroni post hoc ) .

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

Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (n = 3 mice per group, one - way ANOVA, Bonferroni post hoc).


Mean ± CB1 mRNA in cerebellar extracts from WT ASM - KO mice after two months of vehicle or PF treatment ( * * PWTVeh Vs KO Veh = 0 . 0026 , * PKO Veh Vs KO PF = 0 . 0345 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) .

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

Mean ± SEM CB1 mRNA levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (**PWTVeh Vs KO Veh = 0.0026, *PKO Veh Vs KO PF = 0.0345), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).


Immunofluorescence against CB1 in Purkinje of cerebellum of WT ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± associated to CB1 , expressed as of values in vehicle - treated mice ( n = 4 mice per group , > 20 Purkinje per

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

Immunofluorescence against CB1 in Purkinje cells of the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM intensity associated to CB1, expressed as percentage of values in vehicle-treated mice (n = 4 mice per group, > 20 Purkinje cells per mouse


Immunofluorescence against CB1 Lamp1 in Purkinje layer of cerebellum of WT ASM - KO mice after two months of vehicle or PF treatment . Graphs show Mander ´ s coefficient for colocalization of CB1 with Lamp1 ( left ) mean ± lysosomal ( right ) ( * * * PWT Veh vs . KO Veh = 0 . 0002 ; * * PKO Veh vs . KO PF = 0 . 0052 , n = 4 mice per group , > 20 lysosomes per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 5 μm .

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

Immunofluorescence against CB1 and Lamp1 in the Purkinje cell layer of the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graphs show Mander´s coefficient for the colocalization of CB1 with Lamp1 (left) and mean ± SEM lysosomal size (right) (***PWT Veh vs. KO Veh = 0.0002; **PKO Veh vs. KO PF = 0.0052, n = 4 mice per group, >20 lysosomes per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 5 μm.


Immunofluorescence against Purkinje marker Calbindin in cerebellum of WT ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± number of calbindin positive per ( * * * PWTVeh Vs KO Veh < 0 . 0001 , * * PKO PF Vs KO Veh = 0 . 00642 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 100 μm . Immunofluorescence against astrocytic marker GFAP in cerebellum of WT ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± associated to GFAP in arbitrary units ( * * * PWTVeh Vs KO Veh < 0 . 0001 , * * PKO PF Vs KO Veh = 0 . 0083 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm . Immunofluorescence against microglia marker Iba1 in cerebellum of WT ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± number ( left ) ( right ) of Iba1 - positive ( Left * * * PWTVeh Vs KO Veh < 0 . 0001 , * * PKO PF Vs KO Veh = 0 . 0034 ; Right * * * PWTVeh Vs KO Veh < 0 . 0001 , PKO PF Vs KO Veh = 0 . 00123 ) , n = 4 mice per group , > 20 analyzed per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm .

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

Immunofluorescence against the Purkinje cell marker Calbindin in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM number of calbindin positive cells per area (***PWTVeh Vs KO Veh <0.0001, **PKO PF Vs KO Veh = 0.00642), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Scale bar, 100 μm. Immunofluorescence against the astrocytic marker GFAP in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM intensity associated to GFAP in arbitrary units (***PWTVeh Vs KO Veh < 0.0001, **PKO PF Vs KO Veh = 0.0083), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm. Immunofluorescence against the microglia marker Iba1 in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM number (left) and area (right) of Iba1-positive cells (Left ***PWTVeh Vs KO Veh < 0.0001, **PKO PF Vs KO Veh = 0.0034; Right ***PWTVeh Vs KO Veh < 0.0001, PKO PF Vs KO Veh = 0.00123), n = 4 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.


H & E of cerebellum , hippocampus , cortex liver from WT ASM - KO mice 48 hours after one single administration of vehicle or indicated doses of PF . Scale bar , 100 μm .

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

H&E staining of cerebellum, hippocampus, cortex and liver from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. Scale bar, 100 μm.


Mean ± SM , expressed as of vehicle values , in cerebellar , hippocampal , cortical liver extracts from WT ASM - KO mice 48 hours after one single administration of vehicle or indicated doses of PF . ( * PKOVeh Vs KO 5 = 0 . 0304 , n = 3 mice per group , one - way ANOVA , Bonferroni post hoc )

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

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cerebellar, hippocampal, cortical and liver extracts from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. (*PKOVeh Vs KO 5 = 0.0304, n = 3 mice per group, one - way ANOVA, Bonferroni post hoc)


Immunofluorescences against microglia marker Iba1 in cerebellum , hippocampus , cortex or against macrophage marker F4 / 80 in liver from WT ASM - KO mice 48 hours after one single administration of vehicle or indicated doses of PF . Graphs show mean ± number of microglia per unit in cerebellum , hippocampus cortex or mean ± of macrophages in liver ( Hippocampus * * PKOVeh Vs KO 5 = 0 . 0015 , Cortex * PKOVeh Vs KO 5 = 0 . 0364 , Liver * PKOVeh Vs KO 5 = 0 . 0152 , n = 6 mice per group , > 20 analyzed per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm .

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

Immunofluorescences against the microglia marker Iba1 in the cerebellum, hippocampus, cortex or against the macrophage marker F4/80 in the liver from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. Graphs show mean ± SEM number of microglia per area unit in cerebellum, hippocampus and cortex or mean ± SEM area of macrophages in the liver (Hippocampus **PKOVeh Vs KO 5 = 0.0015, Cortex *PKOVeh Vs KO 5 = 0.0364, Liver *PKOVeh Vs KO 5 = 0.0152, n = 6 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.


Western blot against CB1 GAPDH ( used as loading control ) graph showing mean ± CB1 in cerebellar extracts from WT Npc1nmf164 mice ( n = 6 ) .

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

Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts from WT and Npc1nmf164 mice (n = 6).


Immunofluorescence against CB1 Calbindin in cerebellum of WT Npc1nmf164 mice . DAPI stains nuclei . Graphs show mean ± associated to CB1 in Purkinje ( indicated by arrows ) expressed in arbitrary units ( * * P = 0 . 0085 , n = 5 mice per group , Student ' s t - ) . Scale bar , 10 μm .

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

Immunofluorescence images against CB1 and Calbindin in the cerebellum of WT and Npc1nmf164 mice. DAPI stains cell nuclei. Graphs show mean ± SEM intensity associated to CB1 in the Purkinje cells (indicated by arrows) expressed in arbitrary units (**P= 0.0085, n = 5 mice per group, Student's t-test). Scale bar, 10 μm.


Immunofluorescence against CB1 in Purkinje of cerebelllum of - matched control NPC affected children . Graph shows mean ± associated to CB1 in Purkinje expressed as of control values ( 16 57 in control NPC , respectively ) . Scale bar , 10 μm .

B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O B-TISSUE O O O O B-DISEASE O B-ORGANISM O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-DISEASE O O O O O O O O O O

Immunofluorescence images against CB1 in Purkinje cells of the cerebelllum of age-matched control and NPC affected children. Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the control values (16 and 57 cells in control and NPC, respectively). Scale bar, 10 μm.


Graph shows mean ± SM , expressed as of vehicle values , in cultured fibroblasts from a NPC treated with vehicle or with PF at 50μM or 100μM ( n = 2 different cultures )

O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE O O B-DISEASE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

Graph shows mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured fibroblasts from a NPC patient treated with vehicle or with PF at 50μM or 100μM (n= 2 different cultures)


Graph shows mean ± cholesterol , expressed as of vehicle values , in cultured fibroblasts from a NPC treated with vehicle or with PF at 50μM or 100μM ( n = 2 different cultures )

Graph shows mean ± SEM cholesterol levels, expressed as percentage of vehicle values, in cultured fibroblasts from a NPC patient treated with vehicle or with PF at 50μM or 100μM (n=2 different cultures)


Immunofluorescence of CB1 Lamp1 in cultured fibroblasts from control subject NPC treated with vehicle or with PF at 100μM . Graph shows mean ± Mander ´ s coefficient for colocalization of CB1 with Lamp1 ( n = 2 different cultures ) . Scale bar , 5 μm .

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

Immunofluorescence images of CB1 and Lamp1 in cultured fibroblasts from control subject and NPC patient treated with vehicle or with PF at 100μM. Graph shows mean ± SEM Mander´s coefficient for the colocalization of CB1 with Lamp1 (n= 2 different cultures). Scale bar, 5 μm.


Graphs show mean ± SM cholesterol expressed as of values obtained in vehicle treated mice in cerebellum of Npc1nmf164 after 48 hours of a single dose of 5mg / kg PF ( n = 3 mice )

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

Graphs show mean ± SEM SM and cholesterol levels expressed as percentage of the values obtained in the vehicle treated mice in the cerebellum of Npc1nmf164 after 48 hours of a single dose of 5mg/kg PF (n=3 mice)


Immunofluorescence against microglia marker F4 / 80 in cerebellum of WT Npc1nmf164 after 48 hours of a single dose of 5mg / kg PF . Graphs show mean ± number ( upper ) ( lower ) of F4 / 80 positive ( upper graph * * * * P < 0 . 0001 ; lower graph * * * PWT Veh vs NPC Veh = 0 . 0008 ; * PWT Veh vs . NPC PF = 0 . 0286 ; * * * * PWT PF vs . NPC Veh < 0 . 0001 ; * * PWT PF vs . NPC PF = 0 . 0022 , n = 3 mice per group , > 20 analyzed per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm .

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

Immunofluorescence images against the microglia marker F4/80 in the cerebellum of WT and Npc1nmf164 after 48 hours of a single dose of 5mg/kg PF. Graphs show mean ± SEM number (upper) and area (lower) of F4/80 positive cells (upper graph ****P< 0.0001; lower graph ***PWT Veh vs NPC Veh = 0.0008; *PWT Veh vs. NPC PF = 0.0286; ****PWT PF vs. NPC Veh < 0.0001; **PWT PF vs. NPC PF = 0.0022, n = 3 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.


HT - 29 were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . viability was assessed by CellTiter - Glo ( A ) after 16 hours . Data are means ± of three independent experiments . * P < 0 . 1 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cell viability was assessed by CellTiter-Glo (A) after 16 hours. Data are means ± SEM of three independent experiments. *P< 0.1, ****P<0.0001 (ANOVA).


HT - 29 were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . viability was assessed by Crystal Violet ( B ) after 16 hours . Data are means ± of three independent experiments . * P < 0 . 1 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cell viability was assessed by Crystal Violet (B) after 16 hours. Data are means ± SEM of three independent experiments. *P< 0.1, ****P<0.0001 (ANOVA).


HT - 29 were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . Flow cytometric of stained with TO - PRO - 3 nd Annexin V ( A5 ) . Histograms show means ± of three independent experiments . * * * P < 0 . 001 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Flow cytometric analysis of cells stained with TO-PRO-3 nd Annexin V (A5). Histograms show means ± SEM of three independent experiments. ***P<0.001, ****P<0.0001 (ANOVA).


HT - 29 were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . Flow cytometric of stained with Annexin V ( A5 ) . Histograms show means ± of three independent experiments . * * * P < 0 . 001 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Flow cytometric analysis of cells stained with Annexin V (A5). Histograms show means ± SEM of three independent experiments. ***P<0.001, ****P<0.0001 (ANOVA).


HT - 29 were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . Flow cytometric of stained with TO - PRO - 3 Histograms show means ± of three independent experiments . * * * P < 0 . 001 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Flow cytometric analysis of cells stained with TO-PRO-3 Histograms show means ± SEM of three independent experiments. ***P<0.001, ****P<0.0001 (ANOVA).


HT - 29 were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . were collected after 4 24 hours of treatment , stained with TO - PRO - 3 Data are means ± of three independent experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 and 24 hours of treatment, and stained with TO-PRO-3 Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).


HT - 29 were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . were collected after 4 hours of treatment , stained with propidium iodide ( PI ) A5 Data are means ± of three independent experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 hours of treatment, and stained with propidium iodide (PI) and A5 Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).


HT - 29 were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . were collected after 4 24 hours of treatment , stained with propidium iodide ( PI ) Data are means ± of three independent experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 and 24 hours of treatment, and stained with propidium iodide (PI) Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).


HT - 29 were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . J Measurement of lactate deshydrogenase ( LDH ) released from necroptotic , as indicated ( means ± , n = 4 biological replicates , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). J Measurement of lactate deshydrogenase (LDH) released from necroptotic cells, as indicated (means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA).


HT - 29 were transfected with siRNA for MLKL or scramble non - specific ( NS ) siRNA for 72 hours . pre - treated with 10 μM QVD - OPh ( Q ) , 5 μM Birinapant ( S ) , 20 μM necrostatin - 1 ( Nec - 1s ) were stimulated with 1 - 10 ng . mL - 1 TNFα ( T ) . K Time lapsed microscopy of A5 TO - PRO - 3 upon induction of necroptosis . presented data are representative of at least three independent experiments .

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

HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). K Time lapsed microscopy analysis of A5 and TO-PRO-3 staining upon induction of necroptosis. The presented data are representative of at least three independent experiments.


A HT - 29 were transfected with two siRNA for PANX1 , or scramble non - specific ( NS ) siRNA for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) together with 5 μM Birinapant ( S ) , prior stimulation with 10 ng . mL - 1 of TNFα ( T ) , as indicated . Western blotting for hallmarks of necroptosis , as indicated . arrowhead indicates MLKL fragment . Molecular markers ( Mr ) are shown .

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

A HT-29 cells were transfected with two individual siRNA for PANX1, or scramble non-specific (NS) siRNA for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) together with 5 μM Birinapant (S), prior stimulation with 10 ng.mL-1 of TNFα (T), as indicated. Western blotting for hallmarks of necroptosis, as indicated. The arrowhead indicates MLKL cleaved fragment. Molecular weight markers (Mr) are shown.


B were exposed to TQS for 5 hours . Necrostatin - 1 ( Nec - 1s , 20 μM ) was also used . MLKL oligomers ( MLKLn ) were resolved by non - reducing SDS - PAGE after cross - linking .

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

B Cells were exposed to TQS for 5 hours. Necrostatin-1 (Nec-1s, 20 μM) was also used. MLKL oligomers (MLKLn) were resolved by non-reducing SDS-PAGE after cross-linking.


C viability was assessed by CellTiter - Glo after 24 hours of treatment ( means ± , n = 4 biological replicates ) .

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

C The cell viability was assessed by CellTiter-Glo after 24 hours of treatment (means ± SEM, n=4 biological replicates).


Flow cytometric of treated with TQS for 4 hours stained with Annexin V ( D ) Data are means ± of three independent experiments . * P < 0 . 1 , * * * * P < 0 . 0001 ( ANOVA ) .

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

Flow cytometric analysis of cells treated with TQS for 4 hours and stained with Annexin V (D) Data are means ± SEM of three independent experiments. *P<0.1, ****P<0.0001 (ANOVA).


Flow cytometric of treated with TQS for 4 hours stained with TO - PRO - 3 ( E F ) . Data are means ± of three independent experiments . * P < 0 . 1 , * * * * P < 0 . 0001 ( ANOVA ) .

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

Flow cytometric analysis of cells treated with TQS for 4 hours and stained with TO-PRO-3 (E and F). Data are means ± SEM of three independent experiments. *P<0.1, ****P<0.0001 (ANOVA).


HT - 29 were infected with lentiviruses containing two sgRNA to deplete endogenous PANX1 . A control nonspecific ( NC ) was also used . were treated with addition of 1 μM cycloheximide ( C ) to enhance , analyzed by Western blotting ( G ) .

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

HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated with the addition of 1 μM cycloheximide (C) to enhance cell death, and analyzed by Western blotting (G).


HT - 29 were infected with lentiviruses containing two sgRNA to deplete endogenous PANX1 . A control nonspecific ( NC ) was also used . were treated with addition of 1 μM cycloheximide ( C ) to enhance survival was assessed by CellTiter - Glo after 24 hours of treatment ( H ) . Histogram shows means ± , n = 4 biological replicates , * * * * P < 0 . 0001 , ANOVA .

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

HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated with the addition of 1 μM cycloheximide (C) to enhance cell death Cell survival was assessed by CellTiter-Glo after 24 hours of treatment (H). Histogram shows means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA.


HT - 29 were infected with lentiviruses containing two sgRNA to deplete endogenous PANX1 . A control nonspecific ( NC ) was also used . were treated as in ( A ) with addition of 1 μM cycloheximide ( C ) to enhance Annexin V was analyzed by flow cytometry after 4 hours of stimulation Shown are means ± , n = 3 biological replicates , * P < 0 . 1 , * * * * P < 0 . 0001 , ANOVA .

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

HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated as in (A) with the addition of 1 μM cycloheximide (C) to enhance cell death Annexin V staining was analyzed by flow cytometry after 4 hours of stimulation Shown are means ± SEM, n=3 biological replicates, *P<0.1, ****P<0.0001, ANOVA.


HT - 29 were infected with lentiviruses containing two sgRNA to deplete endogenous PANX1 . A control nonspecific ( NC ) was also used . were treated with addition of 1 μM cycloheximide ( C ) to enhance TO - PRO - 3 uptake was analyzed by flow cytometry after 4 hours of stimulation Shown are means ± , n = 3 biological replicates , * P < 0 . 1 , * * * * P < 0 . 0001 , ANOVA .

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

HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated with the addition of 1 μM cycloheximide (C) to enhance cell death TO-PRO-3 uptake was analyzed by flow cytometry after 4 hours of stimulation Shown are means ± SEM, n=3 biological replicates, *P<0.1, ****P<0.0001, ANOVA.


PANX1 expression was restored in PANX1 knockout after an infection with a lentivirus containing a PANX1 cDNA expressing GFP . lysates were analyzed by Western blotting as indicated ( K ) .

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

PANX1 expression was restored in PANX1 knockout cells after an infection with a lentivirus containing a PANX1 cDNA and expressing GFP. Cell lysates were analyzed by Western blotting as indicated (K).


PANX1 expression was restored in PANX1 knockout after an infection with a lentivirus containing a PANX1 cDNA expressing GFP . TO - PRO - 3 uptake was analyzed by flow cytometry Shown are means ± , n = 3 biological replicates , * * * * P < 0 . 0001 ANOVA .

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

PANX1 expression was restored in PANX1 knockout cells after an infection with a lentivirus containing a PANX1 cDNA and expressing GFP. TO-PRO-3 uptake was analyzed by flow cytometry Shown are means ± SEM, n=3 biological replicates, ****P<0.0001 ANOVA.


NS - MLKL - silenced were pretreated with 100 μM carbenoxolone ( CBX ) , 2 mM Probenecid ( Probe ) or 30 μM Trovafloxacin ( Trova ) stimulated survival was evaluated by CellTiter - Glo after 24 hours ( N ) . Data are means ± , n = 4 biological replicates , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ANOVA .

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

NS- and MLKL-silenced cells were pretreated with 100 μM carbenoxolone (CBX), 2 mM Probenecid (Probe) or 30 μM Trovafloxacin (Trova) and stimulated Cell survival was evaluated by CellTiter-Glo after 24 hours (N). Data are means ± SEM, n=4 biological replicates, ***P<0.001, ****P<0.0001, ANOVA.


NS - MLKL - silenced were pretreated with 100 μM carbenoxolone ( CBX ) , 2 mM Probenecid ( Probe ) or 30 μM Trovafloxacin ( Trova ) stimulated TO - PRO - 3 uptake was analyzed by flow cytometry ( O ) . Data are means ± , n = 3 biological replicates , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ANOVA .

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

NS- and MLKL-silenced cells were pretreated with 100 μM carbenoxolone (CBX), 2 mM Probenecid (Probe) or 30 μM Trovafloxacin (Trova) and stimulated TO-PRO-3 uptake was analyzed by flow cytometry (O). Data are means ± SEM, n=3 biological replicates, ***P<0.001, ****P<0.0001, ANOVA.


P were analyzed by Western blotting . presented data are representative of at least three independent experiments .

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

P Cells were analyzed by Western blotting. The presented data are representative of at least three independent experiments.


HT - 29 were transfected with a nonspecific ( NS ) siRNA or with indicated siRNAs for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , exposed to 10 ng . mL - 1 of TNFα ( T ) . NS - MLKL - silenced were treated with 5 μM necrosulfonamide ( NSA ) . MLKL oligomers ( MLKLn ) were visualized by non - reducing SDS - PAGE after cross - linking ( A ) . Data are means ± of three independent experiments . * * * * P < 0 . 0001 ( ANOVA ) . Data information Arrowhead , MLKL fragment .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). NS- and MLKL-silenced cells were treated with 5 μM necrosulfonamide (NSA). MLKL oligomers (MLKLn) were visualized by non-reducing SDS-PAGE after cross-linking proteins (A). Data are means ± SEM of three independent experiments. ****P<0.0001 (ANOVA). Data information Arrowhead, MLKL cleaved fragment.


HT - 29 were transfected with a nonspecific ( NS ) siRNA or with indicated siRNAs for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , exposed to 10 ng . mL - 1 of TNFα ( T ) . NS - MLKL - silenced were treated with 5 μM necrosulfonamide ( NSA ) . MLKL oligomers ( MLKLn ) were exposed to TQS for 5 hours . Molecular markers ( Mr ) are shown . TO - PRO - 3 uptake was analyzed by flow cytometry in treated with TQS for 4 hours ( B ) . Data are means ± of three independent experiments . * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). NS- and MLKL-silenced cells were treated with 5 μM necrosulfonamide (NSA). MLKL oligomers (MLKLn) Cells were exposed to TQS for 5 hours. Molecular weight markers (Mr) are shown. TO-PRO-3 uptake was analyzed by flow cytometry in cells treated with TQS for 4 hours (B). Data are means ± SEM of three independent experiments. ****P<0.0001 (ANOVA).


HT - 29 were transfected with a nonspecific ( NS ) siRNA or with indicated siRNAs for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , exposed to 10 ng . mL - 1 of TNFα ( T ) . C lysates from NS - , ITPK1 - silenced were analyzed by Western blotting as indicated . Data information : Arrowhead , MLKL fragment .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). C Cell lysates from NS-, and ITPK1-silenced cells were analyzed by Western blotting as indicated. Data information: Arrowhead, MLKL cleaved fragment.


HT - 29 were transfected with a nonspecific ( NS ) siRNA or with indicated siRNAs for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , exposed to 10 ng . mL - 1 of TNFα ( T ) . D Western blotting of MLKL

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). D Western blotting analysis of MLKL oligomerization


HT - 29 were transfected with a nonspecific ( NS ) siRNA or with indicated siRNAs for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , exposed to 10 ng . mL - 1 of TNFα ( T ) . E survival was evaluated by CellTiter - Glo after 24 hours of treatment ( means ± , n = 4 biological replicates , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). E Cell survival was evaluated by CellTiter-Glo after 24 hours of treatment (means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA).


HT - 29 were transfected with a nonspecific ( NS ) siRNA or with indicated siRNAs for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , exposed to 10 ng . mL - 1 of TNFα ( T ) . F Measurement of TO - PRO - 3 uptake as in ( B ) . Dead TO - PRO - 3high were discarded . Data are means ± of three independent experiments . * P < 0 . 1 , * * * * P < 0 . 0001 ( ANOVA ) .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). F Measurement of TO-PRO-3 uptake as in (B). Dead TO-PRO-3high cells were discarded. Data are means ± SEM of three independent experiments. *P<0.1, ****P<0.0001 (ANOVA).


HT - 29 were transfected with a nonspecific ( NS ) siRNA or with indicated siRNAs for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , exposed to 10 ng . mL - 1 of TNFα ( T ) . G Small EVs fractions were purified from culture medium by ultracentrifugation . Shown is a representative diagram of particle obtained with tunable resistive sensing ( TRPS , qNano , IZON ) .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). G Small EVs fractions were purified from the culture medium by ultracentrifugation. Shown is a representative diagram of particle count obtained with tunable resistive pulse sensing analysis (TRPS, qNano, IZON).


HT - 29 were transfected with a nonspecific ( NS ) siRNA or with indicated siRNAs for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , exposed to 10 ng . mL - 1 of TNFα ( T ) . H were subjected to Western blotting Data information : Arrowhead , MLKL fragment .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). H Cells were subjected to Western blotting analysis Data information: Arrowhead, MLKL cleaved fragment.


HT - 29 were transfected with a nonspecific ( NS ) siRNA or with indicated siRNAs for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , exposed to 10 ng . mL - 1 of TNFα ( T ) . I MLKL oligomerization was determined Data information Arrowhead , MLKL fragment .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). I MLKL oligomerization was determined Data information Arrowhead, MLKL cleaved fragment.


HT - 29 were transfected with a nonspecific ( NS ) siRNA or with indicated siRNAs for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , exposed to 10 ng . mL - 1 of TNFα ( T ) . J survival by CellTiter - Glo after 24 hours ( mean ± , n = 4 biological replicates , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). J Cell survival by CellTiter-Glo after 24 hours (mean ± SEM, n=4 biological replicates, **P<0.01, ***P<0.001, ****P<0.0001, ANOVA).


HT - 29 were transfected with a nonspecific ( NS ) siRNA or with indicated siRNAs for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , exposed to 10 ng . mL - 1 of TNFα ( T ) . K TO - PRO - 3 uptake Data are means ± , n = 3 biological replicates , * * * * P < 0 . 0001 ( ANOVA ) . presented data are representative of at least three independent experiments .

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

HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). K TO-PRO-3 uptake Data are means ± SEM, n=3 biological replicates, ****P<0.0001 (ANOVA). The presented data are representative of at least three independent experiments.


A HT - 29 were transfected with a siRNA for PANX1 ( # 3 ) , or scramble non - specific ( NS ) siRNA for 72 hours . were pre - treated with 10 μM QVD - OPh ( Q ) plus 5 μM Birinapant ( S ) , exposed to 10 ng . mL - 1 of TNFα ( T ) for 6 hours . Shown is a normalized densitometric for presence of 120 cytokines with an antibody array . color scale ( 0 - 10 ) represents means of normalized densitometry values ( n = 2 technical replicates ) .

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

A HT-29 cells were transfected with a siRNA for PANX1 (#3), or scramble non-specific (NS) siRNA for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T) for 6 hours. Shown is a normalized densitometric analysis for the presence of 120 cytokines with an antibody array. The color scale (0-10) represents the means of normalized densitometry values (n=2 technical replicates).


B , C HT - 29 transfected as indicated ( B ) or PANX1 knockout HT - 29 ( C ) were treated with TQS for 6 hours . mRNA of Cxcl8 relative to untreated NS or NC samples were measured by qPCR ( means ± , n = 3 biological replicates , * * * * P < 0 . 0001 , ANOVA ) .

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

B, C HT-29 transfected as indicated (B) or PANX1 knockout HT-29 cells (C) were treated with TQS for 6 hours. The mRNA levels of Cxcl8 relative to untreated NS or NC samples were measured by qPCR (means ± SEM, n=3 biological replicates, ****P<0.0001, ANOVA).


D Lysates from treated as indicated were prepared subjected to Western blotting as indicated . Molecular markers ( Mr ) are shown . Data information MLKL is indicated with an arrowhead .

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

D Lysates from cells treated as indicated were prepared and subjected to Western blotting analysis as indicated. Molecular weight markers (Mr) are shown. Data information Cleaved MLKL is indicated with an arrowhead.


E Abundance of IL - 8 , measured by ELISA , in supernatants of treated as indicated after 6 hours of stimulation ( means ± , n = 4 biological replicates , * * * P < 0 . 001 , ANOVA ) .

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

E Abundance of IL-8, measured by ELISA, in the supernatants of cells treated as indicated after 6 hours of stimulation (means ± SEM, n=4 biological replicates, ***P<0.001, ANOVA).


HT - 29 were treated as indicated . Cxcl8 mRNA relative to untreated NS samples were measured by qPCR after 6 hours of treatment ( F ) . Data are means ± of three ( F ) independent experiments ( * * * P < 0 . 001 , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were treated as indicated. Cxcl8 mRNA levels relative to untreated NS samples were measured by qPCR after 6 hours of treatment (F). Data are means ± SEM of three (F) independent experiments (***P<0.001, ****P<0.0001, ANOVA).


HT - 29 were treated as indicated . lysates were analyzed by Western blotting ( G ) . Data information : MLKL is indicated with an arrowhead .

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

HT-29 cells were treated as indicated. Cell lysates were analyzed by Western blotting (G). Data information: Cleaved MLKL is indicated with an arrowhead.


HT - 29 were treated as indicated . release of IL - 8 in culture media was analyzed by ELISA ( H ) . Data are means ± of four ( H ) independent experiments ( * * * P < 0 . 001 , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were treated as indicated. The release of IL-8 in cell culture media was analyzed by ELISA (H). Data are means ± SEM of four (H) independent experiments (***P<0.001, ****P<0.0001, ANOVA).


HT - 29 were treated as indicated . Cxcl8 mRNA relative to untreated NS samples were measured by qPCR after 6 hours of treatment ( I ) . Data are means ± of three ( I ) independent experiments ( * * P < 0 . 01 , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were treated as indicated. Cxcl8 mRNA levels relative to untreated NS samples were measured by qPCR after 6 hours of treatment (I). Data are means ± SEM of three (I) independent experiments (**P<0.01, ****P<0.0001, ANOVA).


HT - 29 were treated as indicated . lysates were analyzed by Western blotting ( J ) . Data information : MLKL is indicated with an arrowhead .

HT-29 cells were treated as indicated. Cell lysates were analyzed by Western blotting (J). Data information: Cleaved MLKL is indicated with an arrowhead.


HT - 29 were treated as indicated . IL - 8 release was analyzed in culture media by ELISA ( K ) . Data are means ± of four ( K ) independent experiments ( * * P < 0 . 01 , * * * * P < 0 . 0001 , ANOVA ) .

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

HT-29 cells were treated as indicated. IL-8 release was analyzed in cell culture media by ELISA (K). Data are means ± SEM of four (K) independent experiments (**P<0.01, ****P<0.0001, ANOVA).


A . Experimental demonstration . A1 . Illustration of mapping principle between ChR2 - expressed PV neurons ( green ) regular pyramidal neuron ( Pyr , orange ) . A2 . Acute slices were obtained recorded at ZT0 or ZT12 . A3 . Coronal brain atlas showing visual cortex ( Top left panel , modified from MBL mouse brain atlas ) . Yellow box highlights region of experiment . Top right panel , reference image taken with a 10x objective from a slice prepared from a PV : Ai32 mouse . 0 . 45 mm2 shaded cubic was divided into 15 x 15 grids for independent stimulation . arrowhead indicates position of recording electrode . Bottom panel , representative raw traces of - evoked IPSCs ( LEIPSCs ) recorded from one pyramidal neuron . A4 . spatial distribution of LEIPSCs from example in A3 . A5 . of LEIPSCs in A4 .

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

A. Experimental demonstration. A1. Illustration of the mapping principle between ChR2-expressed PV neurons (green) and regular pyramidal neuron (Pyr, orange). A2. Acute slices were obtained and recorded at ZT0 or ZT12. A3. Coronal brain atlas showing the primary visual cortex (Top left panel, modified from MBL mouse brain atlas). Yellow box highlights the region of experiment. Top right panel, the reference image taken with a 10x objective from a slice prepared from a PV:Ai32 mouse. The 0.45 mm2 shaded cubic area was divided into 15 x 15 grids for independent light stimulation. The white arrowhead indicates the position of the recording electrode. Bottom panel, representative raw traces of light-evoked IPSCs (LEIPSCs) recorded from one pyramidal neuron. A4. The spatial distribution of the LEIPSCs from the example in A3. A5. The heatmap of the LEIPSCs in A4.


B . average heatmaps at ZT0 ZT12 after soma alignment . Soma position was indicated by blue arrow head . C . Comparison of cumulative distribution ( Kolmogorov - Smirnov [ K - S ] ; P = 0 . 002 ) mean ( Inset . ZT0 : 813 . 3 ± 53 . 13 pA ; ZT12 : 1306 ± 93 . 73 pA ; P < 0 . 0001 ; Mann - Whitney U ) of max LEIPSC amplitude between ZT0 ZT12 . D . Comparison of cumulative distribution ( K - S , P = 0 . 0016 ) mean ( Inset . ZT0 : 10 . 44 ± 1 . 44 nA ; ZT12 : 18 . 87 ± 1 . 44 nA ; P < 0 . 0001 ; Mann - Whitney U ) of sum LEIPSC amplitude between ZT0 ZT12 . Data information : Sample sizes used in this figure : ZT0 : 48 from 9 mice ; ZT12 : 51 from 9 mice . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The average heatmaps at ZT0 and ZT12 after soma alignment. Soma position was indicated by the blue arrow head. C. Comparison of the cumulative distribution (Kolmogorov-Smirnov test [K-S test]; P = 0.002) and the mean (Inset. ZT0: 813.3 ± 53.13 pA; ZT12: 1306 ± 93.73 pA; P < 0.0001; Mann-Whitney U test) of the max LEIPSC amplitude between ZT0 and ZT12. D. Comparison of the cumulative distribution (K-S test, P = 0.0016) and the mean (Inset. ZT0: 10.44 ± 1.44 nA; ZT12: 18.87 ± 1.44 nA; P < 0.0001; Mann-Whitney U test) of the sum LEIPSC amplitude between ZT0 and ZT12. Data information: Sample sizes used in this figure: ZT0: 48 cells from 9 mice; ZT12: 51 cells from 9 mice. Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


E . average heatmaps of LEIPSCs after pia aligned to reference image in A3 . blue overlay traces on side of map depicted marginal distribution of sum LEIPSCs for ZT0 ( blue ) ZT12 ( blue ) . F . Comparison of cumulative distribution ( K - S , P = 0 . 0021 ) mean ( Inset , ZT0 : 0 . 067 ± 0 . 004 mm2 ; ZT12 : 0 . 089 ± 0 . 0036 mm2 ; P = 0 . 0003 ; Mann - Whitney U ) inhibitory input between ZT0 ZT12 . Data information : Sample sizes used in this figure : ZT0 : 48 from 9 mice ; ZT12 : 51 from 9 mice . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

E. The average heatmaps of LEIPSCs after pia aligned to the reference image in A3. Dark and light blue overlay traces on the side of the map depicted the marginal distribution of the sum LEIPSCs for ZT0 (Dark blue) and ZT12 (Light blue). F. Comparison of the cumulative distribution (K-S test, P = 0.0021) and the mean (Inset, ZT0: 0.067 ± 0.004 mm2; ZT12: 0.089 ± 0.0036 mm2; P = 0.0003; Mann-Whitney U test) inhibitory input size between ZT0 and ZT12. Data information: Sample sizes used in this figure: ZT0: 48 cells from 9 mice; ZT12: 51 cells from 9 mice. Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


A . mEPSC frequency was higher at ZT12 than ZT0 . Top : example traces of mEPSC recordings . Bottom left : mEPSC frequency ( ZT0 : 12 . 17 ± 0 . 94Hz ; ZT12 : 17 . 03 ± 1 . 20Hz ; P = 0 . 0036 ; Mann - Whitney U ) . Bottom right : cumulative probability histogram of inter - event intervals ( K - S , P < 0 . 0001 ) . B . mIPSC frequency was lower at ZT12 than ZT0 . Top : example traces . Bottom left : mIPSC frequency ( ZT0 : 19 . 15 ± 0 . 99Hz ; ZT12 : 15 . 19 ± 0 . 63Hz ; P = 0 . 0042 ; Mann - Whitney U ) . Bottom right : cumulative probability histogram of inter - event intervals of mIPSCs ( K - S , P < 0 . 0001 ) . C . mEPSC amplitude kinetics did not differ between ZT0 ZT12 . Top : average traces of well - isolated events . Bottom left : mEPSC amplitude ( ZT0 : 20 . 51 ± 0 . 78pA ; ZT12 : 21 . 26 ± 0 . 75pA ; P = 0 . 34 ; Mann - Whitney U ) . Bottom right : distribution of mEPSC amplitude of events ( K - S , P < 0 . 01 ) . D . mIPSC amplitude kinetics did not differ between ZT0 ZT12 . Top : average traces of well - isolated mIPSCs . Bottom left : mIPSC amplitude ( ZT0 : 41 . 31 ± 1 . 91pA ; ZT12 : 44 . 39 ± 1 . 31pA ; P = 0 . 35 ; Mann - Whitney U ) . Bottom right : cumulative distribution of mIPSC amplitudes ( K - S , P < 0 . 0001 Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 .

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

A. mEPSC frequency was higher at ZT12 than ZT0. Top: example traces of mEPSC recordings. Bottom left: mEPSC frequency (ZT0: 12.17 ± 0.94Hz; ZT12: 17.03 ± 1.20Hz; P = 0.0036; Mann-Whitney U test). Bottom right: cumulative probability histogram of the inter-event intervals (K-S test, P < 0.0001). B. mIPSC frequency was lower at ZT12 than ZT0. Top: example traces. Bottom left: mIPSC frequency (ZT0: 19.15 ± 0.99Hz; ZT12: 15.19 ± 0.63Hz; P = 0.0042; Mann-Whitney U test). Bottom right: cumulative probability histogram of the inter-event intervals of mIPSCs (K-S test, P < 0.0001). C. mEPSC amplitude and kinetics did not differ between ZT0 and ZT12. Top: average traces of the well-isolated events. Bottom left: mEPSC amplitude (ZT0: 20.51 ± 0.78pA; ZT12: 21.26 ± 0.75pA; P = 0.34; Mann-Whitney U test). Bottom right: The distribution of mEPSC amplitude of all events (K-S test, P < 0.01). D. mIPSC amplitude and kinetics did not differ between ZT0 and ZT12. Top: average traces of the well-isolated mIPSCs. Bottom left: mIPSC amplitude (ZT0: 41.31 ± 1.91pA; ZT12: 44.39 ± 1.31pA; P = 0.35; Mann-Whitney U test). Bottom right: The cumulative distribution of mIPSC amplitudes (K-S test, P < 0.0001 Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01.


B . Charges of sEPSCs recorded at phase were higher compared to those at phase ( ZT0 : 4 . 21 ± 0 . 28 nC ; ZT4 : 7 . 05 ± 0 . 61 nC ; ZT8 : 7 . 74 ± 0 . 63 nC ; ZT12 : 7 . 81 ± 0 . 59 nC ; ZT18 : 5 . 25 ± 0 . 53 nC ; One - way ANOVA , F ( 4 , 88 ) = 8 . 78 , P < 0 . 0001 , Holm - Sidak post - hoc ) . A copy of 24 - h sEPSCs was drawn with a dashed to demonstrate daily rhythm . C . Charges of sIPSCs oscillate during / cycle ( ZT0 : 45 . 73 ± 2 . 99 nC ; ZT4 : 28 . 85 ± 1 . 76 nC ; ZT8 : 28 . 25 ± 2 . 77 nC ; ZT12 : 28 . 04 ± 2 . 15 nC ; ZT18 : 43 . 37 ± 2 . 42 nC ; One - way ANOVA , F ( 4 , 106 ) = 13 . 65 , P < 0 . 0001 , Holm - Sidak post - hoc ) . A copy of 24 - h sEPSCs was drawn with a dashed to demonstrate daily rhythm . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

B. Charges of sEPSCs recorded at light phase were higher compared to those at dark phase (ZT0: 4.21 ± 0.28 nC; ZT4: 7.05 ± 0.61 nC; ZT8: 7.74 ± 0.63 nC; ZT12: 7.81 ± 0.59 nC; ZT18: 5.25 ± 0.53 nC; One-way ANOVA, F(4,88) = 8.78, P <0.0001, Holm-Sidak post-hoc analysis). A copy of the 24-h sEPSCs was drawn with a dashed line to demonstrate the daily rhythm. C. Charges of sIPSCs oscillate during the light/dark cycle (ZT0: 45.73 ± 2.99 nC; ZT4: 28.85 ± 1.76 nC; ZT8: 28.25 ± 2.77 nC; ZT12: 28.04 ± 2.15 nC; ZT18: 43.37 ± 2.42 nC; One-way ANOVA, F(4,106) = 13.65, P <0.0001, Holm-Sidak post-hoc analysis). A copy of the 24-h sEPSCs was drawn with a dashed line to demonstrate the daily rhythm. Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


E . exposure prevented increase in sEPSCs that normally occurs at ZT12 ( ZT12 as reported in B ; - ZT12 : 5 . 46 ± 0 . 43 nC ; P = 0 . 0082 ; Mann - Whitney U ) . F . exposure did not significantly affect reduction of sIPSCs ( ZT12 as reported in C ; - ZT12 : 33 . 43 ± 1 . 74 nC ; P = 0 . 07 ; Mann - Whitney U ) . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

E. Dark exposure prevented the increase in sEPSCs that normally occurs at ZT12 (ZT12 as reported in B; Dark-ZT12: 5.46 ± 0.43 nC; P = 0.0082; Mann-Whitney U test). F. Dark exposure did not significantly affect the reduction of sIPSCs (ZT12 as reported in C; Dark-ZT12: 33.43 ± 1.74 nC; P = 0.07; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


H . Sleep deprivation further increased sEPSCs compared to ad lib sleep mice ( ZT4 as reported in B ; SD - ZT4 : 11 . 22 ± 0 . 92 nC ; P = 0 . 0013 ; Mann - Whitney U ) . I . Sleep deprivation abolished reduction of sIPSC ( ZT4 as reported in C ; SD - ZT4 : 47 . 13 ± 3 . 39 nC ; P < 0 . 0001 ; Mann - Whitney U ) . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

H. Sleep deprivation further increased sEPSCs compared to ad lib sleep mice (ZT4 as reported in B; SD-ZT4: 11.22 ± 0.92 nC; P = 0.0013; Mann-Whitney U test). I. Sleep deprivation abolished the reduction of sIPSC (ZT4 as reported in C; SD-ZT4: 47.13 ± 3.39 nC; P < 0.0001; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


B - E . Slices were obtained at ZT12 ( B - C ) or ZT0 ( D - E ) pre - incubated with 50 µM CARB for more than 1h before recording mIPSCs in presence of drug . Slices from same were used as controls . CARB increased mIPSC frequency at ZT12 ( CTL : 7 . 74 ± 0 . 55 Hz ; CARB : 12 . 41 ± 1 . 03 Hz ; P = 0 . 0001 ; Mann - Whitney U ) , but not ZT0 ( CTL : 11 . 79 ± 1 . 00 Hz ; CARB : 11 . 47 ± 1 . 60 Hz ; P = 0 . 39 ; Mann - Whitney U ) . mIPSC amplitude was unchanged at either time ( C , ZT12 : CTL : 40 . 94 ± 2 . 08 pA ; CARB : 40 . 64 ± 1 . 66 pA ; P = 0 . 99 ; E , ZT0 : CTL : 34 . 73 ± 2 . 14 pA ; CARB : 33 . 61 ± 2 . 39 pA ; P = 0 . 85 ; Mann - Whitney U ) . Data information : Sample is indicated as ( mice , ) . Error bars in figures indicate standard error of mean ( s . e . m ) . level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY 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 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-E. Slices were obtained at ZT12 (B-C) or ZT0 (D-E) and pre-incubated with 50 µM CARB for more than 1h before recording mIPSCs in the presence of drug. Slices from the same animals were used as controls. CARB increased mIPSC frequency at ZT12 (CTL: 7.74 ± 0.55 Hz; CARB: 12.41 ± 1.03 Hz; P = 0.0001; Mann-Whitney U test), but not ZT0 (CTL:11.79 ± 1.00 Hz; CARB: 11.47 ± 1.60 Hz; P = 0.39; Mann-Whitney U test). mIPSC amplitude was unchanged at either time (C, ZT12: CTL: 40.94 ± 2.08 pA; CARB: 40.64 ± 1.66 pA; P = 0.99; E, ZT0: CTL: 34.73 ± 2.14 pA; CARB: 33.61 ± 2.39 pA; P = 0.85; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.