C . Most of SARS - CoV - 2 - positive ( AB4 , green ) are TUNEL - positive ( i ) some of SARS - CoV - 2 - positive are caspase - positive ( ii ) . Caspase - positive in SARS - CoV - 2 - exposed organoids display pT231Tau localization at soma specified by AT - 180 ( ii ) , which are not observed in in mock organoids ( iii ) . Arrowheads point SARS - CoV - 2 - positive ( AB4 , green ) that are also positive for TUNNEL ( red ) , caspase 3 ( yellow ) Tau AT - 180 ( magenta ) . Figures display scale bars . Bar diagrams at right quantifies proportions of TUNEL caspase - positive among SARS - CoV - 2 - positive . second graph below quantifies proportions of pT231Tau - positive among caspase positive between control exposed groups . At least 400 from 4 organoids two independent ( n = 2 ) batches were examined . Unpaired t , * * P < 0 . 01 . Data presented as mean + . TUNEL - positive in control un - exposed organoids do not contain pT231Tau ( iii ) . Figures display scale bars . Bar diagrams at right quantifies proportions of pT231Tau - positive among TUNNEL - positive between control exposed groups . At least 350 from 4 organoids two independent ( n = 2 ) batches were examined . Unpaired t , * * * P < 0 . 001 . Data presented as mean + .

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

C. Most of the SARS-CoV-2-positive cells (AB4, green) are TUNEL-positive (i) and some of the SARS-CoV-2-positive cells are caspase-positive (ii). Caspase-positive cells in SARS-CoV-2-exposed organoids display pT231Tau localization at the cell soma specified by AT-180 (ii), which are not observed in in mock organoids (iii). Arrowheads point SARS-CoV-2-positive cells (AB4, green) that are also positive for TUNNEL (red), caspase 3 (yellow) and Tau AT-180 (magenta). Figures display scale bars. Bar diagrams at right quantifies proportions of TUNEL and caspase-positive cells among SARS-CoV-2-positive cells. The second graph below quantifies the proportions of pT231Tau-positive cells among caspase positive cells between control and virus exposed groups. At least 400 cells from 4 organoids and two independent (n=2) batches were examined. Unpaired t test, **P<0.01. Data presented as mean + SEM. TUNEL-positive cells in control un-exposed organoids do not contain pT231Tau (iii). Figures display scale bars. Bar diagrams at right quantifies proportions of pT231Tau-positive cells among TUNNEL-positive cells between control and virus exposed groups. At least 350 cells from 4 organoids and two independent (n=2) batches were examined. Unpaired t test, ***P<0.001. Data presented as mean + SEM.


( B ) HA - ATG9A - BirA * was expressed in HCT - 116 ATG13 WT , ATG13 KO or ATG13 KO reconstituted with WT ATG13 or ATG13 ∆ HORMA . were grown in full DMEM media , treated with 50 µM biotin for 12 hrs , followed by detergent lysis incubation with streptavidin resin . graph on right shows of normalized ATG101 infrared signal . Mean ± , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA followed by Fisher ' s LSD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HA-ATG9A-BirA* was expressed in HCT-116 ATG13 WT, ATG13 KO or ATG13 KO cells reconstituted with WT ATG13 or ATG13 ∆HORMA. Cells were grown in full DMEM media, treated with 50 µM biotin for 12 hrs, followed by detergent lysis and incubation with streptavidin resin. The graph on right shows quantification of normalized ATG101 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests.


( C ) were treated as in panel B but included reconstitution with ATG13 ∆ 2AA mutant . graph on right shows of normalized ATG101 infrared signal . Mean ± , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA followed by Fisher ' s LSD . ( right ) .

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

(C) Cells were treated as in panel B but included reconstitution with ATG13 ∆2AA mutant. The graph on right shows quantification of normalized ATG101 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests. (right).


( D ) HA - ATG9A - BirA * was overexpressed in WT ULK1 / 2 Double KO MEFs . were subjected to streptavidin pulldown immunoblotting with indicated antibodies . graph on right shows of normalized ATG13 infrared signal . Mean ± . n = 3 ( biological replicates ) . Significance measured using one - sample t - compared to hypothetical mean of 1 ( right ) . ns = p > 0 . 05 , * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , * * * = p ≤ 0 . 001 , * * * * = p ≤ 0 . 0001 .

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

(D) HA-ATG9A-BirA* was overexpressed in WT and ULK1/2 Double KO MEFs. Cells were subjected to streptavidin pulldown and immunoblotting with indicated antibodies. The graph on right shows quantification of normalized ATG13 infrared signal. Mean ± SEM. n=3 (biological replicates). Significance measured using one-sample t-test compared to hypothetical mean of 1 (right). ns=p>0.05, *=p≤0.05, **= p≤0.01, ***=p≤0.001, ****= p≤0.0001.


( A ) - ( G ) Representative of ATG9A colocalization with different organelle autophagy markers . HCT - 116 ATG9A - HA KI - ATG13 WT or ATG13 KO were grown in full DMEM media , fixed labeled with antibodies for HA indicated organelle / markers imaged ( Scaler bar = 10 µm ) . ( H ) of ATG9A colocalization with indicated organelle markers of golgi ( GOLGIN97 ) , mitochondria ( TUFM ) , ER ( PDIA3 ) , endosomal system ( VPS26A ) , early endosome ( EEA1 ) , lysosome ( LAMP1 ) , autophagy adaptor ( p62 ) , autophagosome markers ( ATG16L1 , WIPI4 , ATG2A , LC3II , WIPI2 ) respectively . Mean ± , n = 1 independent experiment with 30 technical replicates . for p62 colocalization ( G ) are from same single independent

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)-(G) Representative images of ATG9A colocalization with different organelle and autophagy markers. HCT-116 ATG9A-HA KI-ATG13 WT or ATG13 KO cells were grown in full DMEM media, fixed and labeled with antibodies for HA and indicated organelle/cellular markers and imaged (Scaler bar=10 µm). (H) Quantification of ATG9A colocalization with indicated organelle markers of golgi (GOLGIN97), mitochondria (TUFM), ER (PDIA3), endosomal system (VPS26A), early endosome (EEA1), lysosome (LAMP1), autophagy adaptor (p62), and autophagosome markers (ATG16L1, WIPI4, ATG2A, LC3II, WIPI2) respectively. Mean ± SEM, n=1 independent experiment with 30 technical replicates. Images for p62 colocalization (G) are from the same single independent experiment


( A ) Endogenous p62 / SQSTM1 level in HCT - 116 ATG9A - HA KI - ATG13 WT , ATG13 KO , ATG101 KO , ATG9A KO FIP200 KO clones was measured by immunoblotting with indicated antibodies . were grown in full DMEM media , treated with or without 100 nM Bafilomycin for 24 hrs whole lysates were subjected to immunoblotting ( left ) . graph on right shows of normalized p62 infrared signal . Mean ± , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA followed by Fisher ' s LSD ( right ) . ( B ) Endogenous p62 / SQSTM1 level in HEK - 293T ATG13 WT , ATG13 KO , ATG101 KO , ATG9A KO FIP200 KO clones was measured by immunoblotting with indicated . were grown in full DMEM media , treated with or without 100 nM Bafilomycin for 24 hrs whole lysates were subjected to immunoblotting . Mean ± , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA followed by Fisher ' s LSD ( right ) .

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

(A) Endogenous p62/SQSTM1 level in HCT-116 ATG9A-HA KI-ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated antibodies. Cells were grown in full DMEM media, treated with or without 100 nM Bafilomycin for 24 hrs and whole cell lysates were subjected to immunoblotting (left). The graph on right shows quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (B) Endogenous p62/SQSTM1 level in HEK-293T ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated proteins. Cells were grown in full DMEM media, treated with or without 100 nM Bafilomycin for 24 hrs and whole cell lysates were subjected to immunoblotting. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right).


( C ) Confocal of ATG9A colocalization with p62 / SQSTM1 . HCT - 116 ATG9 - HA KI - ATG13 WT , ATG13 KO , ATG101 KO , ATG9A KO FIP200 KO were grown in full DMEM media , fixed , labeled with antibodies for HA p62 / SQSTM1 imaged ( Scale bar 10 µm ) . ATG101 KO was stained quantified separately . are from same single independent experiment ( D ) of average surface of p62 / SQSTM1 in C . Mean ± , n = 1 independent experiment with 30 technical replicates . ATG101 KO was stained quantified separately . Mean ± , n = 1 independent experiment with 30 technical replicates . A break was inserted in Y axis to accommodate wide range of p62 / SQSTM1 sizes . ( E ) of ATG9A colocalization with p62 / SQSTM1 . Mean ± , n = 1 independent experiment with 30 technical replicates . ATG101 KO was stained quantified separately . Mean ± , n = 1 independent experiment with 30 technical replicates .

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

(C) Confocal images of ATG9A colocalization with p62/SQSTM1. HCT-116 ATG9-HA KI-ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO cells were grown in full DMEM media, fixed, labeled with antibodies for HA and p62/SQSTM1 and imaged (Scale bar 10 µm). ATG101 KO was stained and quantified separately. Images are from the same single independent experiment (D) Quantification of average surface area of p62/SQSTM1 puncta in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates. ATG101 KO was stained and quantified separately. Mean ± SEM, n=1 independent experiment with 30 technical replicates. A break was inserted in the Y axis to accommodate the wide range of p62/SQSTM1 puncta sizes. (E) Quantification of ATG9A colocalization with p62/SQSTM1. Mean ± SEM, n=1 independent experiment with 30 technical replicates. ATG101 KO was stained and quantified separately. Mean ± SEM, n=1 independent experiment with 30 technical replicates.


( A ) Endogenous p62 / SQSTM1 level was measured in HCT - 116 ATG9A - HA KI - ATG13 WT , ATG13 KO OR ATG13 KO reconstituted with ATG13 WT , ATG13 ΔHORMA ATG13 Δ2AA by immunoblotting with indicated ( left ) . were grown in full DMEM media whole lysates were subjected to immunoblotting ( left ) . of normalized p62 infrared signal . Mean ± , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA followed by Fisher ' s LSD ( right ) . ( B ) HCT - 116 in A were incubated in EBSS for 4 hours then processed analyzed as in A .

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

(A) Endogenous p62/SQSTM1 level was measured in HCT-116 ATG9A-HA KI-ATG13 WT, ATG13 KO OR ATG13 KO cells reconstituted with ATG13 WT, ATG13 ΔHORMA and ATG13 Δ2AA by immunoblotting with indicated proteins (left). Cells were grown in full DMEM media and whole cell lysates were subjected to immunoblotting (left). Quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (B) HCT-116 cells in A were incubated in EBSS for 4 hours then processed and analyzed as in A.


( C ) Confocal of ATG9A colocalization with p62 / SQSTM1 in HCT - 116 ATG9A - HA KI - ATG13 WT , ATG13 KO or ATG13 KO reconstituted with ATG13 WT , ATG13 Δ2AA ATG13 ΔHORMA . were grown in full DMEM media , fixed , labelled with antibodies for HA p62 / SQSTM1 imaged ( Scale bar = 10 µm ) . are from same single independent experiment ( D ) of average surface of p62 / SQSTM1 in C . Mean ± , n = 1 independent experiment with 30 technical replicates . A break was inserted in Y axis to accommodate wide range of p62 / SQSTM1 sizes . ( E ) of ATG9A colocalization with p62 / SQSTM1 in C . Mean ± , n = 1 independent experiment with 30 technical replicates .

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

(C) Confocal images of ATG9A colocalization with p62/SQSTM1 in HCT-116 ATG9A-HA KI- ATG13 WT, ATG13 KO or ATG13 KO cells reconstituted with ATG13 WT, ATG13 Δ2AA and ATG13 ΔHORMA. Cells were grown in full DMEM media, fixed, labelled with antibodies for HA and p62/SQSTM1 and imaged (Scale bar=10 µm). Images are from the same single independent experiment (D) Quantification of average surface area of p62/SQSTM1 puncta in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates. A break was inserted in the Y axis to accommodate the wide range of p62/SQSTM1 puncta sizes. (E) Quantification of ATG9A colocalization with p62/SQSTM1 in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates.


( B ) Confocal of Ubiquitin colocalization with p62 / SQSTM1 in HCT116 ATG13 WT HCT116 ATG13 KO . were grown in full DMEM media , fixed , labelled with antibodies for Ubiquitin p62 / SQSTM1 imaged ( Scale bar = 10 µm ) ( left ) . of Ubiquitin colocalization with p62 / SQSTM1 ( right ) . Mean ± , n = 1 independent experiment with 30 technical replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images of Ubiquitin colocalization with p62/SQSTM1 in HCT116 ATG13 WT and HCT116 ATG13 KO cells. Cells were grown in full DMEM media, fixed, labelled with antibodies for Ubiquitin and p62/SQSTM1 and imaged (Scale bar=10 µm) (left). Quantification of Ubiquitin colocalization with p62/SQSTM1 (right). Mean ± SEM, n=1 independent experiment with 30 technical replicates.


( C ) Modified chase experiment . HEK293T ATG13 WT HEK293T ATG13 KO stably expressing HA ATG9A - BirA * were labeled with biotin for 24 hours , media was replaced with full DMEM streptavidin pulldown was performed at indicated time points . of normalized p62 infrared signal . Mean ± , n = 3 ( biological replicates ) . Significance measured using Student ' s t ( bottom ) . ns = p > 0 . 05 , * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , * * * = p ≤ 0 . 001 , * * * * = p ≤ 0 . 0001 .

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

(C) Modified pulse chase experiment. HEK293T ATG13 WT and HEK293T ATG13 KO cells stably expressing HA ATG9A-BirA* were pulse labeled with biotin for 24 hours, media was replaced with full DMEM and streptavidin pulldown was performed at indicated time points. Quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using Student's t test (bottom). ns=p>0.05, *=p≤0.05, **=p≤0.01, ***=p≤0.001, ****= p≤0.0001.


( C ) Confocal of Venus colocalization with ATG9A ULK1 in HCT116 ATG9A - HA KI ATG13 - ATG101 double KO stably expressing VenusC - ATG101 - 3X FLAG VenusN - ATG13 - Myc or VenusN - ATG13 Δ2AA - Myc . were grown in full DMEM media , fixed , labelled with antibodies for HA ULK1 imaged ( Scale bar = 10 µm ) . of overlap shown as of total Venus ( bottom ) . Mean ± , n = 1 independent experiment with 10 technical replicates .

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

(C) Confocal images of Venus colocalization with ATG9A and ULK1 in HCT116 ATG9A-HA KI ATG13-ATG101 double KO cells stably expressing VenusC-ATG101-3X FLAG and VenusN-ATG13-Myc or VenusN-ATG13 Δ2AA-Myc. Cells were grown in full DMEM media, fixed, labelled with antibodies for HA and ULK1 and imaged (Scale bar=10 µm). Quantification of overlap shown as percent of total Venus puncta (bottom). Mean ± SEM, n=1 independent experiment with 10 technical replicates.


( E ) Confocal of EGFP - p62 / SQSTM1 colocalization with HA ATG9A ATG13 in HCT116 ATG9A - HA KI stably expressing EGFP - p62 / SQSTM1 . were grown in full DMEM media with or without 1 μM Wortmannin for time 4 hrs , fixed , labelled with antibodies for HA ATG13 imaged ( Scale bar = 10 µm ) .

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

(E) Confocal images of EGFP-p62/SQSTM1 colocalization with HA ATG9A and ATG13 in HCT116 ATG9A-HA KI cells stably expressing EGFP-p62/SQSTM1. Cells were grown in full DMEM media with or without 1 μM Wortmannin for time 4 hrs, fixed, labelled with antibodies for HA and ATG13 and imaged (Scale bar=10 µm).


A - C . Scanning electron microscopy ( ) of ( A ) nanowires ( VNw ; scale - 5 µm ) ( B ) crude nanosheets ( VSh ; scale - 200 nm ) , ( C ) ultrathin - nanosheets ( Vs ) , scale - 20 µm .

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

A-C. Scanning electron microscopy (SEM) images of (A) nanowires (VNw; scale - 5 µm) and (B) crude nanosheets (VSh; scale - 200 nm), (C) ultrathin-nanosheets (Vs), scale - 20 µm.


C . Michaelis - Menten plot with variation of H2O2 ( 0 - 400 µM ) in presence of Vs ( 20 ng / µL ) , GSH ( 2 mM ) , NADPH ( 0 . 2 mM ) , GR ( 1 . 7 units ) in phosphate buffer ( 100 mM , pH 7 . 4 ) at 25 ° C .

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

C. Michaelis-Menten plot with the variation of H2O2 (0-400 µM) in the presence of Vs (20 ng/µL), GSH (2 mM), NADPH (0.2 mM), GR (1.7 units) in phosphate buffer (100 mM, pH 7.4) at 25°C.


D . Michaelis - Menten plot with variation of GSH ( 0 - 7 mM ) in presence of Vs ( 20 ng / μL ) , H2O2 ( 200 µM ) , NADPH ( 0 . 2 mM ) , GR ( 1 . 7 units ) in phosphate buffer ( 100 mM , pH 7 . 4 ) at 25 ° C .

D. Michaelis-Menten plot with the variation of GSH (0-7 mM) in the presence of Vs (20 ng/μL), H2O2 (200 µM), NADPH (0.2 mM), GR (1.7 units) in phosphate buffer (100 mM, pH 7.4) at 25°C.


G , H . U1 Grx1 - roGFP2 were supplemented with BSO ( G ) or GSH ( H ) for 16 h to deplete or replenish GSH , respectively . Following this , were treated with Vs for 15 min , exposed to H2O2 , ratiometric response was measured .

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

G, H. U1 Grx1-roGFP2 cells were supplemented with BSO (G) or GSH (H) for 16 h to deplete or replenish GSH, respectively. Following this, cells were treated with Vs for 15 min, exposed to H2O2, and the ratiometric response was measured.


E . U1 - Grx1 - roGFP2 were serum starved for 30 min in presence or absence of Vs sodium selenite ( 0 . 5 nM ) , biosensor response was measured . Data were compared to serum starved control ( C ) .

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

E. U1-Grx1-roGFP2 cells were serum starved for 30 min in the presence or absence of Vs and sodium selenite (0.5 nM), and the biosensor response was measured. Data were compared to serum starved control cells (C).


F . U1 were either serum - starved or supplemented with Se ( 0 . 5 nM ) or Vs ( 0 . 62 ng / μL ) HIV reactivation was measured at 6 h post starvation by gag RT - PCR .

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

F. U1 cells were either serum-starved or supplemented with Se (0.5 nM) or Vs (0.62 ng/μL) and HIV reactivation was measured at 6 h post starvation by gag RT-PCR.


Total isolated from untreated ( UT ) , PMA - treated , Vs - treated Vs + PMA treated U1 was examined by NanoString technology to assess expression of genes responsive to oxidative stress HIV . ( C Heat map showing functional categories of DEGs under PMA / UT , Vs / UT PMA + Vs / UT comparisons . mRNA were normalised using internal control β2 microglobulin ( B2M ) , fold change ( FC ) was calculated using nSolver 4 . 0 software . Genes showing an absolute FC > 1 . 5 , P < 0 . 05 were considered as significantly altered .

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

Total RNA isolated from untreated (UT), PMA-treated, Vs-treated and Vs+PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV. (C Heat map showing functional categories of DEGs under PMA/UT, Vs/UT and PMA+Vs/UT comparisons. mRNA counts were normalised using the internal control β2 microglobulin (B2M), and fold change (FC) was calculated using the nSolver 4.0 software. Genes showing an absolute FC >1.5, and P <0.05 were considered as significantly altered.


Total isolated from untreated ( UT ) , PMA - treated , Vs - treated Vs + PMA treated U1 was examined by NanoString technology to assess expression of genes responsive to oxidative stress HIV . D ) Heat map showing functional categories of DEGs under PMA / UT , Vs / UT PMA + Vs / UT comparisons . mRNA were normalised using internal control β2 microglobulin ( B2M ) , fold change ( FC ) was calculated using nSolver 4 . 0 software . Genes showing an absolute FC > 1 . 5 , P < 0 . 05 were considered as significantly altered .

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

Total RNA isolated from untreated (UT), PMA-treated, Vs-treated and Vs+PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV. D) Heat map showing functional categories of DEGs under PMA/UT, Vs/UT and PMA+Vs/UT comparisons. mRNA counts were normalised using the internal control β2 microglobulin (B2M), and fold change (FC) was calculated using the nSolver 4.0 software. Genes showing an absolute FC >1.5, and P <0.05 were considered as significantly altered.


B - D . A similar was performed using Jurkat ( CD4 + T ) viral replication was assessed by ( B ) gag RT PCR , ( C ) p24 ELISA in culture supernatant , ( D ) immunoblotting for p24 ( viral capsid ) in whole lysate .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. A similar assay was performed using Jurkat (CD4+ T cell line) and viral replication was assessed by (B) gag RT PCR, (C) p24 ELISA in the culture supernatant, and (D) immunoblotting for p24 (viral capsid protein) in the whole cell lysate.


F . CD4 + T purified from human PBMCs ( 3 healthy donors ) were activated , pre - treated with 25 ng / μL Vs for 15 min , infected with 0 . 05 moi of HIV - 1 NL - 4 . 3 . released in supernatant was quantified by p24 ELISA . Vs treatment was repeated every 48 h .

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F. Primary CD4+ T cells purified from human PBMCs (3 healthy donors) were activated, pre-treated with 25 ng/μL Vs for 15 min, and infected with 0.05 moi of HIV-1 NL-4.3. Virus released in supernatant was quantified by p24 ELISA. Vs treatment was repeated every 48 h.


B . Expanded CD4 + T from 3 were cultured in presence of IL - 2 ARVs , with without 25 ng / µL Vs for 21 days . Vs treatment was given for 15 min every 3rd day . HIV transcripts were quantified by RT - qPCR at day 14 , day 21 , at 24 h post - stimulation of cultured for 21 days by prostratin . Limit of detection for RT - qPCR was 3 viral transcripts per million .

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B. Expanded CD4+ T cells from 3 patients were cultured in presence of IL-2 and ARVs, with and without 25 ng/µL Vs for 21 days. Vs treatment was given for 15 min every 3rd day. HIV transcripts were quantified by RT-qPCR at day 14, day 21, and at 24 h post-stimulation of cells cultured for 21 days by prostratin. Limit of detection for RT-qPCR was 3 viral transcripts per million cells.


C . At day 21 , were stimulated with 1 µM prostratin for 24 h HIV transcripts were quantified by RT - qPCR . Reduction in viral stimulation in Vs treated samples are represented as values . ND - non determined . D . Aggregate plot for 3 from data ( C ) .

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C. At day 21, cells were stimulated with 1 µM prostratin for 24 h and HIV transcripts were quantified by RT-qPCR. Reduction in viral stimulation in Vs treated samples are represented as percentage values. ND - non determined. D. Aggregate plot for 3 patients from data (C).


Mean ± CB2 mRNA in cerebellar extracts of WT ASM - KO mice ( n = 5 mice per group ) .

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Mean ± SEM CB2 mRNA levels in cerebellar extracts of WT and ASM-KO mice (n = 5 mice per group).


Western blot against CB2 PSD - 95 ( used as loading control ) graph showing mean ± CB2 in cerebellar extracts of WT ASM - KO mice ( n = 6 mice per group ) .

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Western blot against CB2 and PSD-95 (used as loading control) and graph showing mean ± SEM CB2 protein levels in cerebellar extracts of WT and ASM-KO mice (n = 6 mice per group).


Mean ± CB1 mRNA in cerebellar extracts of WT ASM - KO mice ( * * P = 0 . 0013 , n = 6 mice per group , Student ' s t - ) .

Mean ± SEM CB1 mRNA levels in cerebellar extracts of WT and ASM-KO mice (**P = 0.0013, n = 6 mice per group, Student's t-test).


Western blot against CB1 GAPDH ( used as loading control ) graph showing mean ± CB1 in cerebellar extracts of WT ASM - KO mice ( n = 3 mice per group ) .

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Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts of WT and ASM-KO mice (n = 3 mice per group).


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 .

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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 .

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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 - ) .

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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 - ) .

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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 .

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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 .

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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 )

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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 ) .

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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 - ) .

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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 - )

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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 .

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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 ) .

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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 - ) .

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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 - ) .

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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 - ) .

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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 .

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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 ) .

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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 ) .

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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 - ) .

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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 ) .

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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 ) .

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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 .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 )

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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 ) .

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

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 ) .

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

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

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

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

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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

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

Western blot 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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 .

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

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

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

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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 ) .

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 ) .

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

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

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 O 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 ) .

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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

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 .

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

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 .

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

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 .

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

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 ) .

O O O O O O O O O O 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. 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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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). 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR 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). 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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). 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O 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). 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O 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). 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 ) .

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

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

B, C 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 O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O 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. Cell lysates were analyzed by Western blotting (G). Data information: Cleaved MLKL is indicated with an arrowhead.