( A - D ) The serum concentrations of S1P ( A ) , apoM ( B ) , albumin ( C ) and HDL - C ( D ) in noICU ( n = 89 ) and ICU ( n = 22 ) patients are shown . The box - plots represent the interquartile range with median ( central line ) ; the whiskers represent the measured range of noICU and ICU patients . Each S1P and apoM measurement was run in triplicate , and performed at least twice in independent assays . Two - tailed Student ' s t - test was used for statistical analysis . * * * * p < 0 . 0001 .

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

(A-D) The serum concentrations of S1P (A), apoM (B), albumin (C) and HDL-C (D) in noICU (n=89) and ICU (n= 22) patients are shown. The box-plots represent the interquartile range with median (central line); the whiskers represent the measured range of noICU and ICU patients. Each S1P and apoM measurement was run in triplicate, and performed at least twice in independent assays. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.


Pearson correlation between S1P ( A - C ) PSI days from admission and NLR in COV ( n = 111 ) . Scatter plots and fitted regression line are shown in each figure . Exact p values or * * * * p < 0 . 0001 are reported .

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

Pearson correlation between S1P (A-C) PSI days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.


Pearson correlation between apoM ( D - F ) and PSI , days from admission and NLR in COV ( n = 111 ) . Scatter plots and fitted regression line are shown in each figure . Exact p values or * * * * p < 0 . 0001 are reported .

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

Pearson correlation between apoM (D-F) and PSI , days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.


( A , B ) Cumulative risk for ICU admission ( A ) and cumulative survival ( B ) in COV patients ( n = 111 ) , grouped for cut - off value of S1P serum level of 0 . 60 μM . Statistical analysis was performed by Cox regression . Exact p values are reported .

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

(A,B) Cumulative risk for ICU admission (A) and cumulative survival (B) in COV patients (n=111), grouped for cut-off value of S1P serum level of 0.60 μM. Statistical analysis was performed by Cox regression. Exact p values are reported.


( b ) Suppression of Htt103Q toxicity in yeast by Gpx1 , Hyr1 ( Gpx3 ) and mGpx1 . The viability of the parental strain , Y258 , and of cells overexpressing yeast GPxs was determined using spotting assays . Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose ( GAL ) to induce the expression of Htt103Q and the indicated ORF .

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

(b) Suppression of Htt103Q toxicity in yeast by Gpx1, Hyr1 (Gpx3) and mGpx1. The viability of the parental strain, Y258, and of cells overexpressing yeast GPxs was determined using spotting assays. Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose (GAL) to induce the expression of Htt103Q and the indicated ORF.


a ) Total GPx activity was measured in mGpx1 - overexpressing ( mGpx1 ) or control ( WT ) cell lines with ( induced ) or without ( uninduced ) Htt103Q expression . Overexpression of mGpx1 increases total GPx activity in PC12 cells ( * * * * P 0 . 0001 compared to uninduced controls ; n = 7 per condition ) .

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

a) Total GPx activity was measured in mGpx1-overexpressing (mGpx1) or control (WT) cell lines with (induced) or without (uninduced) Htt103Q expression. Overexpression of mGpx1 increases total GPx activity in PC12 cells (****P 0.0001 compared to uninduced controls; n = 7 per condition).


( b ) Quantification of caspase - 3 / 7 activation in response to Htt103Q expression in cells overexpressing mGpx1 . Overexpression of mGpx1 reduces Htt103Q - mediated caspase - 3 / 7 activation ( * * * * P 0 . 0001 ) . Values represent the ratio of mGpx1 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q ( n = 12 ( WT ) ; n = 9 ( monomeric red fluorescent protein ( mRFP ) ) ; n = 8 ( mGpx1 ) per condition ) .

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

(b) Quantification of caspase-3/7 activation in response to Htt103Q expression in cells overexpressing mGpx1. Overexpression of mGpx1 reduces Htt103Q-mediated caspase-3/7 activation (****P 0.0001). Values represent the ratio of mGpx1 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q (n = 12 (WT); n = 9 (monomeric red fluorescent protein (mRFP)); n = 8 (mGpx1) per condition).


( c ) Effect of the GPx mimetic ebselen on Htt103Q - mediated caspase activation . A single 10 μM dose of ebselen significantly reduced caspase - 3 / 7 activation in Htt103Q - expressing cells ( * * * * P 0 . 0001 ) . Values represent the ratio of caspase - 3 / 7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q . Statistical analysis was performed using an unpaired , two - tailed Mann - Whitney test ( n = 12 per condition ) .

O O O O O O O O B-CONTROLLED_VAR 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

(c) Effect of the GPx mimetic ebselen on Htt103Q-mediated caspase activation. A single 10 μM dose of ebselen significantly reduced caspase-3/7 activation in Htt103Q-expressing cells (****P 0.0001). Values represent the ratio of caspase-3/7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q. Statistical analysis was performed using an unpaired, two-tailed Mann-Whitney test (n = 12 per condition).


( d ) mGpx1 overexpression ( * * * * P 0 . 0001 ) and ebselen treatment ( * * * * P 0 . 0001 ) significantly reduce Htt103Q - mediated ROS production ( n = 9 per condition ) . Unless stated otherwise , all statistical analyses were performed using one - way analysis of variance ( ANOVA ) with post - hoc tests . NS , not significant . * * P 0 . 01 . All data are shown as the mean ± s . e . m .

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

(d) mGpx1 overexpression (****P 0.0001) and ebselen treatment (****P 0.0001) significantly reduce Htt103Q-mediated ROS production (n = 9 per condition). Unless stated otherwise, all statistical analyses were performed using one-way analysis of variance (ANOVA) with post-hoc tests. NS, not significant. **P 0.01. All data are shown as the mean ± s.e.m.


( a ) Pseudopupil images from wild - type flies , Huntington ' s disease flies and Huntington ' s disease flies overexpressing mGpx1 at day 10 . In wild - type flies , seven rhabdomeres are visible per ommatidium . Scale bar , ∼ 10

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

(a) Pseudopupil images from wild-type flies, Huntington's disease flies and Huntington's disease flies overexpressing mGpx1 at day 10. In wild-type flies, seven rhabdomeres are visible per ommatidium. Scale bar, ∼10 μM


( b ) Quantification of the average number of rhabdomeres per ommatidium in Huntington ' s disease flies with and without pan - neural mGpx1 overexpression at days 1 ( n = 14 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) , 7 ( n = 7 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) and 10 ( n = 8 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) after eclosion . Significant effects of genotype ( F1 , 50 = 82 . 3 , P = 3 . 82 × 10 − 12 ) and age ( F2 , 50 = 94 . 3 , P = 1 . 06 × 10 − 17 ) were observed , as well as a significant interaction between genotype and age ( F2 , 50 = 7 . 76 , P = 0 . 00116 ) , indicating that mGpx1 reduces the rate of neurodegeneration in Htt93Q flies . Post - hoc Newman - Keuls comparisons showed significant rescue of neurodegeneration in mGpx1 - expressing flies at days 1 ( * P 0 . 05 ) , 7 ( * * * P 0 . 001 ) and 10 ( * * * P 0 . 001 ) . Browne - Forsythe analyses for equality of variance revealed a significant inequality of variance ( P = 0 . 02 ) due to a single group ( Htt93Q + mGpx1 , day 1 ) . The nonparametric Mann - Whitney test confirmed a difference at day 1 between the two groups ( P = 0 . 03 ) , and between Htt93Q + mGpx1 flies at days 1 and 7 ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with and without pan-neural mGpx1 overexpression at days 1 (n = 14 (Htt93Q); n = 9 (Htt93Q + mGpx1)), 7 (n = 7 (Htt93Q); n = 9 (Htt93Q + mGpx1)) and 10 (n = 8 (Htt93Q); n = 9 (Htt93Q + mGpx1)) after eclosion. Significant effects of genotype (F1,50 = 82.3, P = 3.82 × 10−12) and age (F2,50 = 94.3, P = 1.06 × 10−17) were observed, as well as a significant interaction between genotype and age (F2,50 = 7.76, P = 0.00116), indicating that mGpx1 reduces the rate of neurodegeneration in Htt93Q flies. Post-hoc Newman-Keuls comparisons showed significant rescue of neurodegeneration in mGpx1-expressing flies at days 1 (*P 0.05), 7 (***P 0.001) and 10 (***P 0.001). Browne-Forsythe analyses for equality of variance revealed a significant inequality of variance (P = 0.02) due to a single group (Htt93Q + mGpx1, day 1). The nonparametric Mann-Whitney test confirmed a difference at day 1 between the two groups (P = 0.03), and between Htt93Q + mGpx1 flies at days 1 and 7 (P 0.0001).


( c ) Total locomotor activity was assessed as the average number of beam crossings per 30 - min bin per fly . Htt93Qex1 - expressing flies ( n = 29 ) have reduced locomotor activity compared to controls ( * * * P 0 . 001 ; n = 30 ( WT ( w118 ) ) ; n = 32 ( mGpx1 ) ) , which is restored by mGpx1 ( * * P 0 . 01 ; n = 32 ) . Comparisons were performed by Kruskal - Wallis one - way ANOVA .

(c) Total locomotor activity was assessed as the average number of beam crossings per 30-min bin per fly. Htt93Qex1-expressing flies (n = 29) have reduced locomotor activity compared to controls (***P 0.001; n = 30 (WT (w118)); n = 32 (mGpx1)), which is restored by mGpx1 (**P 0.01; n = 32). Comparisons were performed by Kruskal-Wallis one-way ANOVA.


( d ) Neurodegeneration of Pdf clock neurons was assessed using confocal microscopy in adult flies at day 7 . Flies expressing Htt93Qex1 driven by Pdf - GAL4 ( n = 16 brain hemispheres ) showed loss of s - LNVs compared to Canton S ( CS ) control flies ( n = 13 ) , which was ameliorated by coexpression of mGpx1 ( * * * * P 0 . 0001 ; n = 16 ) . Comparisons were performed by Kruskal - Wallis one - way ANOVA .

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

(d) Neurodegeneration of Pdf clock neurons was assessed using confocal microscopy in adult flies at day 7. Flies expressing Htt93Qex1 driven by Pdf-GAL4 (n = 16 brain hemispheres) showed loss of s-LNVs compared to Canton S (CS) control flies (n = 13), which was ameliorated by coexpression of mGpx1 (****P 0.0001; n = 16). Comparisons were performed by Kruskal-Wallis one-way ANOVA.


( e ) Quantification of the average number of rhabdomeres per ommatidium in Huntington ' s disease flies with ebselen or vehicle ( DMSO ; n = 10 ) treatment at day 7 after eclosion and in newly emerged day 0 adult flies ( untreated control ; n = 10 ) . Significant treatment effects were observed , both with the 100 μM ( * * P 0 . 01 ; n = 9 ) and 300 μM ( * * * P 0 . 001 ; n = 9 ) doses of ebselen . All data are shown as the mean ± s . e . m .

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

(e) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with ebselen or vehicle (DMSO; n = 10) treatment at day 7 after eclosion and in newly emerged day 0 adult flies (untreated control; n = 10). Significant treatment effects were observed, both with the 100 μM (**P 0.01; n = 9) and 300 μM (***P 0.001; n = 9) doses of ebselen. All data are shown as the mean ± s.e.m.


PC12 cells were treated with either 10 μM ebselen or 10 mM N - acetylcysteine ( NAc ) for 24 h , with the addition of 400 nM bafilomycin A1 for the final 4 h . Autophagy was induced in the indicated samples by the addition of 200 nM rapamycin for 24 h . ( a ) Autophagy was determined by measuring the amount of LC3 - II by immunoblot analysis . ( b ) Densitometric analysis of the bands was performed using ImageJ , and the amounts of LC3 - II are expressed relative to the untreated control . All statistical analyses were performed using one - way ANOVA with post - hoc tests ( * * * * P 0 . 0001 ) . All data are shown as the mean ± s . e . m . ; n = 7 per condition .

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

PC12 cells were treated with either 10 μM ebselen or 10 mM N-acetylcysteine (NAc) for 24 h, with the addition of 400 nM bafilomycin A1 for the final 4 h. Autophagy was induced in the indicated samples by the addition of 200 nM rapamycin for 24 h. (a) Autophagy was determined by measuring the amount of LC3-II by immunoblot analysis. (b) Densitometric analysis of the bands was performed using ImageJ, and the amounts of LC3-II are expressed relative to the untreated control. All statistical analyses were performed using one-way ANOVA with post-hoc tests (****P 0.0001). All data are shown as the mean ± s.e.m.; n = 7 per condition.


A . BMDMM were treated as indicated with either 60ng / mL ultra - pure ( UP ) LPS , 50ng / mL human derived recombinant GM - CSF , 40µM WEHD - FMK for 16 hours or 5µM nigericin for the last hour . Supernatants were then analysed for IL - 1β secretion by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

A. BMDMM were treated as indicated with either 60ng/mL ultra-pure (UP) LPS, 50ng/mL human derived recombinant GM-CSF, 40µM WEHD-FMK for 16 hours or 5µM nigericin for the last hour. Supernatants were then analysed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.


B . HoxB8 macrophages were treated with the indicated dilutions of GM - CSF using supernatants from GM - CSF expressing cells . Shown are ELISA analysis of IL - 1β secretion in supernatants and western blots indicating IL - 1β expression in lysates . . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HoxB8 macrophages were treated with the indicated dilutions of GM-CSF using supernatants from GM-CSF expressing cells. Shown are ELISA analysis of IL-1β secretion in supernatants and western blots indicating IL-1β expression in lysates.. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.


C . HoxB8 macrophages of the indicated genotypes were treated as indicated with 60ng / mL UP - LPS , 1 % GM - CSF containing supernatant ( approximately 2ng / mL GM - CSF ) , 40µM WEHD . FMK , 0 , 5µM CRT - 0066101 for 16 hours , 5µM nigericin was added for the last hour . Supernatants were analysed by ELISA for IL - 1β . Inlay panels show western blots for levels of IL - 1β expression in the indicated knockouts . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

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

C. HoxB8 macrophages of the indicated genotypes were treated as indicated with 60ng/mL UP-LPS, 1% GM-CSF containing supernatant (approximately 2ng/mL GM-CSF), 40µM WEHD.FMK, 0,5µM CRT-0066101 for 16 hours, 5µM nigericin was added for the last hour. Supernatants were analysed by ELISA for IL-1β. Inlay panels show western blots for levels of IL-1β expression in the indicated knockouts. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.


D . HoxB8 cells were treated with 60ng / mL of UP - LPS , 1 % GM - CSF supernatant , 50mM KCl , and 5µM nigericin as indicated for 16 hours before supernatants were analysed for IL - 1β by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

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 B-CONTROLLED_VAR 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

D. HoxB8 cells were treated with 60ng/mL of UP-LPS, 1% GM-CSF supernatant, 50mM KCl, and 5µM nigericin as indicated for 16 hours before supernatants were analysed for IL-1β by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.


A . BMDMM ( left panel ) and HoxB8 macrophages ( right panel ) were treated as indicated with either LPS or LPS + GM - CSF . Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 . Shown is the densitometric quantification of Nrf2 protein level from three independent experiments . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For the western blot quantification , significance was calculated using unpaired t - test . p values are shown .

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 O O O O O O O O O O O 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. BMDMM (left panel) and HoxB8 macrophages (right panel) were treated as indicated with either LPS or LPS+ GM-CSF. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2. Shown is the densitometric quantification of Nrf2 protein level from three independent experiments. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


B . HoxB8 macrophages were treated as in A . and levels of IL - 1β in the supernatant were analysed by ELISA Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For the western blot quantification , significance was calculated using unpaired t - test . p values are shown .

B. HoxB8 macrophages were treated as in A. and levels of IL-1β in the supernatant were analysed by ELISA Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


C . HoxB8 macrophages were treated for 12 hours with LPS or LPS + GM - CSF and RNA extracted and analysed by qPCR for the indicated targets . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For the western blot quantification , significance was calculated using unpaired t - test . p values are shown .

C. HoxB8 macrophages were treated for 12 hours with LPS or LPS+GM-CSF and RNA extracted and analysed by qPCR for the indicated targets. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


D . Wild type and Tnf - / - HoxB8 cells were treated as indicated . Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 and HMOX1 . For wild type cells , MitoTEMPO ( 50µM ) was also added as indicated . Shown are the densitometric quantifications of NRF2 and HMOX1 protein levels of three independent experiments ( right panel ) . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For the western blot quantification , significance was calculated using unpaired t - test . p values 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 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

D. Wild type and Tnf-/- HoxB8 cells were treated as indicated. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 and HMOX1. For wild type cells, MitoTEMPO (50µM) was also added as indicated. Shown are the densitometric quantifications of NRF2 and HMOX1 protein levels of three independent experiments (right panel). Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


E . HoxB8 cells of the indicated genotypes were treated with LPS for 8 hours followed by staining with mitoSOX red . The fluorescent signal was analyzed using flow cytometry . Shown are average geometric mean fluorescence intensity of mitoSOX red . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For the western blot quantification , significance was calculated using unpaired t - test . p values 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HoxB8 cells of the indicated genotypes were treated with LPS for 8 hours followed by staining with mitoSOX red. The fluorescent signal was analyzed using flow cytometry. Shown are average geometric mean fluorescence intensity of mitoSOX red. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


F . Tnf - / - HoxB8 macrophages were treated with LPS with or without 50ng / ml recombinant TNF for 16 hours . Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 and HMOX1 ( upper panel ) . Shown are the densitometric quantifications of indicated protein levels of three independent experiments ( lower panel ) . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For the western blot quantification , significance was calculated using unpaired t - test . p values 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Tnf-/- HoxB8 macrophages were treated with LPS with or without 50ng/ml recombinant TNF for 16 hours. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 and HMOX1 (upper panel). Shown are the densitometric quantifications of indicated protein levels of three independent experiments (lower panel). Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


A . HoxB8 macrophages were seeded in an 8 - well Ibidi chamber and treated as indicated for 12 hours . Cells were fixed and stained for ASC ( Red ) and with DAPI ( blue ) . For nigericin treatments nigericin was added for only 30 minutes . Example cells containing ASC Specks are shown with white arrows . Scale bars represent 50µM . Data information : Microscopy pictures are representative images from 3 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 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

A. HoxB8 macrophages were seeded in an 8-well Ibidi chamber and treated as indicated for 12 hours. Cells were fixed and stained for ASC (Red) and with DAPI (blue). For nigericin treatments nigericin was added for only 30 minutes. Example cells containing ASC Specks are shown with white arrows. Scale bars represent 50µM. Data information: Microscopy pictures are representative images from 3 biological replicates.


B . HoxB8 cells were treated as indicated with LPS or LPS + GM - CSF . The cross linker DSS was added to the cells as described in methods . Samples were analysed by western blot for ASC oligomerization . Black vertical lines indicate that non - relevant lanes were removed during figure preparation but samples were run on the same gel . Data information : westerns are representative images from 3 biological replicates .

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

B. HoxB8 cells were treated as indicated with LPS or LPS+GM-CSF. The cross linker DSS was added to the cells as described in methods. Samples were analysed by western blot for ASC oligomerization. Black vertical lines indicate that non-relevant lanes were removed during figure preparation but samples were run on the same gel. Data information: westerns are representative images from 3 biological replicates.


C . Wild type and caspase - 1 - / - HoxB8 macrophages were treated as indicated for 16 hours . Cell lysates were made and analysed by western blot for levels of caspase - 1 and gasdermin D cleavage . Data information : westerns are representative images from 3 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 O O O O O 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. Wild type and caspase-1-/- HoxB8 macrophages were treated as indicated for 16 hours. Cell lysates were made and analysed by western blot for levels of caspase-1 and gasdermin D cleavage. Data information: westerns are representative images from 3 biological replicates.


D . HoxB8 macrophages were treated as indicated with LPS or LPS + GM - CSF for 12 hours . Cells were lysed and NLRP3 was immune precipitated using a magnetic bead coupled antibody . As a negative control , beads without NLRP3 antibody were used . Beads were boiled and analysed for recruitment of NLRP3 , and caspase - 1 . * indicates light chain from the antibody . Molecular weight markers shown in middle of NLRP3 blot were embedded in image using Chemostar imaging software ( INTAS ) at time of image acquisition . Data information : westerns are representative images from 3 biological replicates .

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 O O O O O 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. HoxB8 macrophages were treated as indicated with LPS or LPS+GM-CSF for 12 hours. Cells were lysed and NLRP3 was immune precipitated using a magnetic bead coupled antibody. As a negative control, beads without NLRP3 antibody were used. Beads were boiled and analysed for recruitment of NLRP3, and caspase-1. * indicates light chain from the antibody. Molecular weight markers shown in middle of NLRP3 blot were embedded in image using Chemostar imaging software (INTAS) at time of image acquisition. Data information: westerns are representative images from 3 biological replicates.


E . HoxB8 macrophages were incubated with combinations of LPS , GM - CSF , nigericin + / - Z - VAD . FMK as indicated . Proteins were extracted and analysed by western blot for caspase - 1 processing . Data information : westerns are representative images from 3 biological replicates .

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

E. HoxB8 macrophages were incubated with combinations of LPS, GM-CSF, nigericin +/- Z-VAD.FMK as indicated. Proteins were extracted and analysed by western blot for caspase-1 processing. Data information: westerns are representative images from 3 biological replicates.


F . HoxB8 macrophages were seeded in a 96 well microplate and co - treated with either 10µM or 50µM z - VAD with LPS + GM - CSF for 16 hours . Then 100nM of Sytox Green was added to the cells . The number of sytox green + cells per image was quantified . Data information : Significance was calculated using one way ANOVA with multiple comparisons . Every dot represents one biological replicate . P values are shown .

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

F. HoxB8 macrophages were seeded in a 96 well microplate and co-treated with either 10µM or 50µM z-VAD with LPS+GM-CSF for 16 hours. Then 100nM of Sytox Green was added to the cells. The number of sytox green+ cells per image was quantified. Data information: Significance was calculated using one way ANOVA with multiple comparisons. Every dot represents one biological replicate. P values are shown.


A . HoxB8 macrophages were treated for 16 hours with LPS + / - GM - CSF as indicated . Proteins were extracted and analysed for levels of IL - 1β by western blot .

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

A. HoxB8 macrophages were treated for 16 hours with LPS +/- GM-CSF as indicated. Proteins were extracted and analysed for levels of IL-1β by western blot.


B . HoxB8 macrophages were treated with LPS or LPS + GM - CSF as indicated for 16 hours . Cells were lysed in buffer containing FLAG tagged K63 TUBE and K63 ubiquitylated proteins were precipitated using anti FLAG pull down . Pulled down proteins were analysed for IL - 1β and ubiquitin by western blot .

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

B. HoxB8 macrophages were treated with LPS or LPS+GM-CSF as indicated for 16 hours. Cells were lysed in buffer containing FLAG tagged K63 TUBE and K63 ubiquitylated proteins were precipitated using anti FLAG pull down. Pulled down proteins were analysed for IL-1β and ubiquitin by western blot.


C . IL - 1β - / - hoxB8 macrophages were infected with lentivirus expressing IL - 1β - FLAG - 6xHis or the indicated mutants from the IL - 1β promoter . Cells were treated as indicated with LPS or LPS + GM - CSF or LPS + Nigericin and levels of secreted IL - 1β were analysed by ELISA ( top panel ) . GM - CSF treated cells were also lysed in urea buffer and nickel NTA affinity pull down was performed . Nickel NTA pull down was also performed on supernatants ( bottom panel ) . Pulled down proteins form the His tag pull down were analysed for the presence of IL - 1β by western blot . Molecular weight markers shown in middle of blot were embedded in image using Chemostar imaging software ( INTAS ) at time of image acquisition . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values 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 O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. IL-1β-/- hoxB8 macrophages were infected with lentivirus expressing IL-1β-FLAG-6xHis or the indicated mutants from the IL-1β promoter. Cells were treated as indicated with LPS or LPS+GM-CSF or LPS+Nigericin and levels of secreted IL-1β were analysed by ELISA (top panel). GM-CSF treated cells were also lysed in urea buffer and nickel NTA affinity pull down was performed. Nickel NTA pull down was also performed on supernatants (bottom panel). Pulled down proteins form the His tag pull down were analysed for the presence of IL-1β by western blot. Molecular weight markers shown in middle of blot were embedded in image using Chemostar imaging software (INTAS) at time of image acquisition. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


D . HoxB8 macrophages were treated as in A with addition of 50µM mitoTEMPO or 10mM NAC as indicated . Proteins were extracted and analysed for IL - 1β by western blot .

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

D. HoxB8 macrophages were treated as in A with addition of 50µM mitoTEMPO or 10mM NAC as indicated. Proteins were extracted and analysed for IL-1β by western blot.


E . Wild type and TNF - / - HoxB8 macrophages were treated as indicated with LPS or LPS + GM - CSF . Proteins were extracted and analysed for IL - 1β by western blot . Black vertical lines indicate that non - relevant lanes were removed during figure preparation but samples were run on the same gel .

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

E. Wild type and TNF-/- HoxB8 macrophages were treated as indicated with LPS or LPS+GM-CSF. Proteins were extracted and analysed for IL-1β by western blot. Black vertical lines indicate that non-relevant lanes were removed during figure preparation but samples were run on the same gel.


F . HoxB8 macrophages were treated as in A but with the addition of 25µM Punicalagin throughout the 16 hour time course . Proteins were extracted and analysed by western blot ( left panel ) . Supernatants were also analysed by ELISA for secreted IL - 1β ( right panel ) . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values are shown .

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

F. HoxB8 macrophages were treated as in A but with the addition of 25µM Punicalagin throughout the 16 hour time course. Proteins were extracted and analysed by western blot (left panel). Supernatants were also analysed by ELISA for secreted IL-1β (right panel). Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


A . Wild type and GsdmD - / - HoxB8 macrophages were treated as indicated for 16 hours . Supernatants were analyzed for IL - 1β secretion by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Wild type and GsdmD-/- HoxB8 macrophages were treated as indicated for 16 hours. Supernatants were analyzed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


B . HoxB8 macrophages were seeded in an 8 - well Ibidi chamber and treated for 14 hours with LPS and GM - CSF . Cells were fixed and stained for GasderminD ( green ) , IL - 1β ( red ) and DAPI ( blue ) . Images were taken at the Zeiss LSM880 Confocal microscope Data information : shown . Microscopy pictures are representative images from 3 biological replicates . Scale bars in microscopy images represent 10µM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HoxB8 macrophages were seeded in an 8-well Ibidi chamber and treated for 14 hours with LPS and GM-CSF. Cells were fixed and stained for GasderminD (green), IL-1β (red) and DAPI (blue). Images were taken at the Zeiss LSM880 Confocal microscope Data information: shown. Microscopy pictures are representative images from 3 biological replicates. Scale bars in microscopy images represent 10µM.


C . HoxB8 macrophages were co - stimulated with three different PEG 3mM for 16 hours as indicated . Supernatants were analyzed for IL - 1β secretion by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values are shown .

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

C. HoxB8 macrophages were co-stimulated with three different PEG 3mM for 16 hours as indicated. Supernatants were analyzed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


D . HoxB8 macrophages were seeded in 96 well microplate and treated as indicated . Before treatment , sytox green was added and cells were imaged in an incucyte incubator microscope every hour for 16 hours . Shown is the sytox green signal normalized to the total cell area . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HoxB8 macrophages were seeded in 96 well microplate and treated as indicated. Before treatment, sytox green was added and cells were imaged in an incucyte incubator microscope every hour for 16 hours. Shown is the sytox green signal normalized to the total cell area. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


E . Wild type and GsdmD - / - HoxB8 macrophages were treated as indicated for 16 hours . Proteins were extracted and analysed for IL - 1β ubiquitination by western blot . Data information : westerns are representative images from 3 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 O O O O O O O O O O O O O O O O O O O O O

E. Wild type and GsdmD-/- HoxB8 macrophages were treated as indicated for 16 hours. Proteins were extracted and analysed for IL-1β ubiquitination by western blot. Data information: westerns are representative images from 3 biological replicates.


( A ) Family pedigree . Variant segregation analysis of PSMC3 . Electropherogram of a part of intron 10 of PSMC3 encompassing the identified variation ( c . [ 1127 + 337A > G ] ; [ 1127 + 337A > G ] , p . [ ( Ser376Arg15 * ) ] ; [ ( Ser376Arg15 * ) ] ) in the affected individuals , their unaffected parents and siblings . The variation was found at the homozygous state in the affected individuals ( II . 2 , II . 4 , II . 7 ) , at the heterozygous state in the parents ( I . 1 , I . 2 , I . 3 , I . 4 , I . 5 , I . 6 ) and was either at the heterozygous state ( II . 5 ) or absent in the unaffected siblings ( II . 1 , II . 3 , II . 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Family pedigree. Variant segregation analysis of PSMC3. Electropherogram of a part of intron 10 of PSMC3 encompassing the identified variation (c.[1127+337A>G];[1127+337A>G], p.[(Ser376Arg15*)];[(Ser376Arg15*)]) in the affected individuals, their unaffected parents and siblings. The variation was found at the homozygous state in the affected individuals (II.2, II.4, II.7), at the heterozygous state in the parents (I.1, I.2, I.3, I.4, I.5, I.6) and was either at the heterozygous state (II.5) or absent in the unaffected siblings (II.1, II.3, II.6).


( B ) Face ( up ) and profile ( down ) photographs for patients II . 4 ( a : 8 yo , b : 16 yo ) , II . 2 ( c : 6 yo , d : 14 yo ) and II . 7 ( e : 1 yo , f : 7 yo ) over time . One can observe prominent supraciliay arches , synophris , sunken cheeks , short philtrum and retrusion in the malar region .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Face (up) and profile (down) photographs for patients II.4 (a: 8 yo, b: 16 yo), II.2 (c: 6 yo, d: 14 yo) and II.7 (e: 1 yo, f: 7 yo) over time. One can observe prominent supraciliay arches, synophris, sunken cheeks, short philtrum and retrusion in the malar region.


( C ) Sub cutaneous calcifications found only on knees ( g : 9 yo and h : 16 yo ) on elbows ( i : 9 yo ) of patient II . 4 . White hair were present only on the legs of the 3 patients as illustrated for patient II . 4 ( j : 16 yo ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Sub cutaneous calcifications found only on knees (g: 9 yo and h: 16 yo) on elbows (i: 9 yo) of patient II.4. White hair were present only on the legs of the 3 patients as illustrated for patient II.4 (j: 16 yo).


( D ) Temporal bone CT - scan from patient II . 7 ( left column ) and a control ( right column ) showing malformation of the semicircular canal . The left ear is shown on the upper panels while de right ear on the lower panels .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Temporal bone CT-scan from patient II.7 (left column) and a control (right column) showing malformation of the semicircular canal. The left ear is shown on the upper panels while de right ear on the lower panels.


( E ) Amplification of the cDNA fragment between exons 9 - 10 and 11 of PSMC3 showing the abnormally spliced RNA fragment . One band at 180bp representing the normal allele is seen for the control and two bands for the individual II . 4 ( pathologic allele at 300bp ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Amplification of the cDNA fragment between exons 9-10 and 11 of PSMC3 showing the abnormally spliced RNA fragment. One band at 180bp representing the normal allele is seen for the control and two bands for the individual II.4 (pathologic allele at 300bp).


( A ) Anti - ubiquitin western blot in control and patient fibroblasts and total amount of proteins loaded ( stainfree ) showing increased ubiquitination in patient cells ( lane 4 ) .

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

(A) Anti-ubiquitin western blot in control and patient fibroblasts and total amount of proteins loaded (stainfree) showing increased ubiquitination in patient cells (lane 4).


( B ) Histogram showing the quantification of ubiquitin with western blot assays . The data shown correspond to the sum of all bands detected by the anti - ubiquitin antibody expressed as a percentage of the amount of ubiquitin in " Control 1 " cells . Bars show mean of ten independent experiments + / - SD ( n = 10 , t - test * : p < 0 . 01 , * * : p < 0 . 05 ) .

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

(B) Histogram showing the quantification of ubiquitin with western blot assays. The data shown correspond to the sum of all bands detected by the anti-ubiquitin antibody expressed as a percentage of the amount of ubiquitin in "Control 1" cells. Bars show mean of ten independent experiments +/- SD (n=10, t-test *: p<0.01, **: p<0.05).


( C ) Mass spectrometry results from the co immunoprecipitation with PSMC3 are displayed as the normalized total number of spectra count of each protein computed as the mean from 3 controls ( x axis ) vs the mean of patient II . 4 triplicate . Proteasome subcomplexes are colored according to the displayed legend and standard ratio lines are drawn .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mass spectrometry results from the co immunoprecipitation with PSMC3 are displayed as the normalized total number of spectra count of each protein computed as the mean from 3 controls (x axis) vs the mean of patient II.4 triplicate. Proteasome subcomplexes are colored according to the displayed legend and standard ratio lines are drawn.


( A ) Whole - cell lysates from control and patient ( case index , CI ) fibroblasts were assessed by 3 - 12 % native - PAGE gradient gels with proteasome bands ( 30S , 26S and 20S complexes ) visualized by their ability to cleave the Suc - LLVY - AMC fluorogenic peptide .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Whole-cell lysates from control and patient (case index, CI) fibroblasts were assessed by 3-12% native-PAGE gradient gels with proteasome bands (30S, 26S and 20S complexes) visualized by their ability to cleave the Suc-LLVY-AMC fluorogenic peptide.


( B ) Ten micrograms of control and patient cell lysates were tested for their chymotrypsin - like activity by incubating them with 0 . 1 mM of the Suc - LLVY - AMC substrate at 37 ° C over a 180 - h period of time in quadruplicates on a 96 - well plate . Indicated on the y - axis are the raw fluorescence values measured by a microplate reader and reflecting the AMC cleavage from the peptide . Bars show the mean of 4 independent experiments + / - SD .

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

(B) Ten micrograms of control and patient cell lysates were tested for their chymotrypsin-like activity by incubating them with 0.1 mM of the Suc-LLVY-AMC substrate at 37°C over a 180-h period of time in quadruplicates on a 96-well plate. Indicated on the y-axis are the raw fluorescence values measured by a microplate reader and reflecting the AMC cleavage from the peptide. Bars show the mean of 4 independent experiments +/- SD.


( C ) Proteasome complexes from control and patient fibroblasts separated by native - PAGE were subjected to western - blotting using antibodies specific for α6 , Rpt5 ( PSMC3 ) and PA28 - α , as indicated .

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

(C) Proteasome complexes from control and patient fibroblasts separated by native-PAGE were subjected to western-blotting using antibodies specific for α6, Rpt5 (PSMC3) and PA28-α, as indicated.


( D ) Proteins extracted from control and CI PSMC3 were separated by 10 or 12 . 5 % SDS - PAGE prior to western - blotting using primary antibodies directed against ubiquitin and several proteasome subunits and / or components including α6 , β1 , β ; β5 , β5i , Rpt2 ( PSMC1 ) , Rpt5 ( PSMC3 ) , Rpt3 ( PSMC4 ) , Rpt4 ( PSMC6 ) and PA28 - α , as indicated . For the PSMC3 staining , two exposure times are shown . Arrow indicates an additional PSMC3 species corresponding to the expected size of the truncated PSMC3 variant . Equal protein loading between samples was ensured by probing the membrane with an anti - α - Tubulin antibody .

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

(D) Proteins extracted from control and CI PSMC3 were separated by 10 or 12.5% SDS-PAGE prior to western-blotting using primary antibodies directed against ubiquitin and several proteasome subunits and/or components including α6, β1, β; β5, β5i, Rpt2 (PSMC1), Rpt5 (PSMC3), Rpt3 (PSMC4), Rpt4 (PSMC6) and PA28-α, as indicated. For the PSMC3 staining, two exposure times are shown. Arrow indicates an additional PSMC3 species corresponding to the expected size of the truncated PSMC3 variant. Equal protein loading between samples was ensured by probing the membrane with an anti-α-Tubulin antibody.


Control and patient ( index case , IC PSMC3 ) fibroblasts were exposed to a 16 - h treatment with 30 nM of the proteasome inhibitor carfilzomib or left untreated ( as a negative control ) . Following treatment , cells were collected and subjected to RIPA - mediated protein extraction prior to SDS - PAGE and subsequent western - blotting using antibodies specific for ubiquitin , TCF11 / Nrf1 , Rpt1 ( PSMC2 ) , Rpt3 ( PSMC4 ) , Rpt5 ( PSMC3 ) , Rpt6 ( PSMC5 ) , β1 , β2 , β5 , β5i , β1i , PA28 - α and α - Tubulin ( loading control ) as indicated . For the TCF11 / Nrf1 staining , two exposure times 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Control and patient (index case, IC PSMC3) fibroblasts were exposed to a 16-h treatment with 30 nM of the proteasome inhibitor carfilzomib or left untreated (as a negative control). Following treatment, cells were collected and subjected to RIPA-mediated protein extraction prior to SDS-PAGE and subsequent western-blotting using antibodies specific for ubiquitin, TCF11/Nrf1, Rpt1 (PSMC2), Rpt3 (PSMC4), Rpt5 (PSMC3), Rpt6 (PSMC5), β1, β2, β5, β5i, β1i, PA28-α and α-Tubulin (loading control) as indicated. For the TCF11/Nrf1 staining, two exposure times are shown.


( A ) Control and / or patient ( index case , IC PSMC3 ) fibroblasts were subjected to a 24 - h transfection with pcDNA3 . 1 / empty vector ( mock ) or pcDNA3 . 1 / PSMC3 prior to RIPA - mediated protein extraction and subsequent western - blotting using antibodies specific for ubiquitin , PSMC3 ( i . e . Rpt5 ) and α - Tubulin ( loading control ) .

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

(A) Control and/or patient (index case, IC PSMC3) fibroblasts were subjected to a 24-h transfection with pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3 prior to RIPA-mediated protein extraction and subsequent western-blotting using antibodies specific for ubiquitin, PSMC3 (i.e. Rpt5) and α-Tubulin (loading control).


( B ) Densitometry analysis showing the relative ubiquitin contents detected by western blotting in patient fibroblasts exposed to either pcDNA3 . 1 / empty vector ( mock ) or pcDNA3 . 1 / PSMC3 , as indicated . The y - axis represents the percent changes in densitometry measurements ( of pixel intensities using Image J ) which are set as 100 % for cells transfected with the pcDNA3 . 1 / empty vector ( mock ) at 24 post - transfection ( n = 4 , * : p < 0 . 05 , t - test ) . Bars show the mean + / - SEM .

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

(B) Densitometry analysis showing the relative ubiquitin contents detected by western blotting in patient fibroblasts exposed to either pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3, as indicated. The y-axis represents the percent changes in densitometry measurements (of pixel intensities using Image J) which are set as 100 % for cells transfected with the pcDNA3.1/empty vector (mock) at 24 post-transfection (n=4, *: p<0.05, t-test). Bars show the mean +/- SEM.


( C ) Patient ( index case , IC PSMC3 ) fibroblasts transfected with either pcDNA3 . 1 / empty vector ( mock ) or pcDNA3 . 1 / PSMC3 were exposed to a 30 - nM treatment of carfilzomib or left untreated ( as a negative control ) . After 16 hours , cells were collected and subjected to RIPA - mediated protein extraction prior to SDS - PAGE and subsequent western - blotting using antibodies specific for ubiquitin , TCF11 / Nrf1 , Rpt1 ( PSMC2 ) , Rpt3 ( PSMC4 ) , Rpt5 ( PSMC3 ) , Rpt6 ( PSMC5 ) , β1 , β2 , β5 , β5i , PA28 - α and α - Tubulin ( loading control ) , as indicated . For the TCF11 / Nrf1 staining , two exposure times 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Patient (index case, IC PSMC3) fibroblasts transfected with either pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3 were exposed to a 30-nM treatment of carfilzomib or left untreated (as a negative control). After 16 hours, cells were collected and subjected to RIPA-mediated protein extraction prior to SDS-PAGE and subsequent western-blotting using antibodies specific for ubiquitin, TCF11/Nrf1, Rpt1 (PSMC2), Rpt3 (PSMC4), Rpt5 (PSMC3), Rpt6 (PSMC5), β1, β2, β5, β5i, PA28-α and α-Tubulin (loading control), as indicated. For the TCF11/Nrf1 staining, two exposure times are shown.


( B - B ' ) . Cataract detection revealed abnormal lens reflection in psmc3 morpholino ( MO ) - mediated knockdown but not in controls ( uninj , ctrl - mo ) . Similarly , abnormal lens reflection was also observed in embryos injected with sgRNA + Cas9 but not in sgRNA injected embryos without Cas9 ( sgRNA2 ) . Co - injection of wt psmc3 mRNA with either psmc3 - mo or sgRNA2 + Cas9 reduced the number of embryos presenting abnormal lens reflection . Scale bar = 50 µm . ( B ' ) Quantification of embryos with abnormal lens reflection .

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

(B-B'). Cataract detection revealed abnormal lens reflection in psmc3 morpholino (MO)-mediated knockdown but not in controls (uninj, ctrl-mo). Similarly, abnormal lens reflection was also observed in embryos injected with sgRNA + Cas9 but not in sgRNA injected embryos without Cas9 (sgRNA2). Co-injection of wt psmc3 mRNA with either psmc3-mo or sgRNA2 + Cas9 reduced the number of embryos presenting abnormal lens reflection. Scale bar = 50 µm. (B') Quantification of embryos with abnormal lens reflection.


( D - D ' ) bright field images of inner ear development ( lateral position ) . ( D ) Epithelial projections were fused and formed canal pillars in 4 - day - old uninjected and control injected fish ( ctrl - mo , sgRNA2 ) but not in morphants ( mo ) and crispants ( sgRNA2 + Cas9 ) . Co - injection of wt psmc3 mRNA with psmc3 - mo or sgRNA + Cas9 reduced the number of embryos presenting abnormal ear phenotype . Black asterisks indicate fused pillars . Red arrowheads mark unfused projections . Scale bar = 100 µm . ( D ' ) Quantification of embryos with abnormal projection outgrowth .

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

(D-D') bright field images of inner ear development (lateral position). (D) Epithelial projections were fused and formed canal pillars in 4-day-old uninjected and control injected fish (ctrl-mo, sgRNA2) but not in morphants (mo) and crispants (sgRNA2+Cas9). Co-injection of wt psmc3 mRNA with psmc3-mo or sgRNA + Cas9 reduced the number of embryos presenting abnormal ear phenotype. Black asterisks indicate fused pillars. Red arrowheads mark unfused projections. Scale bar = 100 µm. (D') Quantification of embryos with abnormal projection outgrowth.


( E - E ' ) An anti - acetylated tubulin antibody ( green ) staining revealed an abnormal amount of kinocilia in psmc3 crispants ( sgRNA2 + Cas9 ) compared to uninjected and control injected embryos ( sgRNA2 ) . Nuclei are stained in blue with DAPI . Representative images show kinocilia of the lateral cristae . Scale bar = 20 µm . ( E ' ) Quantification of embryos with an abnormal amount of kinocilia .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E') An anti-acetylated tubulin antibody (green) staining revealed an abnormal amount of kinocilia in psmc3 crispants (sgRNA2+Cas9) compared to uninjected and control injected embryos (sgRNA2). Nuclei are stained in blue with DAPI. Representative images show kinocilia of the lateral cristae. Scale bar = 20 µm. (E') Quantification of embryos with an abnormal amount of kinocilia.


High and constitutive expression of CXCR4 in the membrane of SW1417 CRC cells as measured by flow cytometry .

High and constitutive expression of CXCR4 in the membrane of SW1417 CRC cells as measured by flow cytometry.


Lack of human SDF - 1α release from cultured SW1417 CRC cells , as measured by ELISA , whereas human control 1BR3 . G fibroblasts express high SDF - 1α levels , after 48h or 72h of growth in culture . ( N = 2 experiment in duplicate )

Lack of human SDF-1α release from cultured SW1417 CRC cells, as measured by ELISA, whereas human control 1BR3.G fibroblasts express high SDF-1α levels, after 48h or 72h of growth in culture. (N=2 experiment in duplicate)


Nanoconjugate internalization in CXCR4 overexpressing ( CXCR4 + ) SW1417 CRC cells after 1 hour exposure at 1µM , as measured by fluorescence emission using flow cytometry . ( N = 3 experiments in duplicate ) Significant difference at p = 0 . 002 between T22 - GFP - H6 - FdU and AMD3100 + T22 - GFP - H6 - FdU

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

Nanoconjugate internalization in CXCR4 overexpressing (CXCR4+) SW1417 CRC cells after 1 hour exposure at 1µM, as measured by fluorescence emission using flow cytometry. (N=3 experiments in duplicate) Significant difference at p= 0.002 between T22-GFP-H6-FdU and AMD3100+T22-GFP-H6-FdU groups


Intracellular trafficking of T22 - GFP - H6 - FdU in CXCR4 + SW1417 cells by confocal microscopy after exposure at 1µM for 24 h . The green staining corresponds to GFP containing nanoconjugates and the red staining corresponds to plasma cell membranes stained with a red dye ( CellMask ™ ) , whereas cell nucleus was stained in blue with hoescht . The insets show detail of the intracellular localization of nanostructured , fluorescent entities , in an isosurface representation within a three - dimensional volumetric x - y - z data

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

Intracellular trafficking of T22-GFP-H6-FdU in CXCR4+ SW1417 cells by confocal microscopy after exposure at 1µM for 24 h. The green staining corresponds to GFP containing nanoconjugates and the red staining corresponds to plasma cell membranes stained with a red dye (CellMask™), whereas cell nucleus was stained in blue with hoescht. The insets show detail of the intracellular localization of nanostructured, fluorescent entities, in an isosurface representation within a three-dimensional volumetric x-y-z data field


Linearized T22 - GFP - H6 - FdU dose - response trend line representation compared with unconjugated free oligo - FdU exposure . Antitumor effect was measured as CXCR4 + SW1417 cell viability by MTT after 72 h exposure as the described concentrations . ( N = 3 experiments in duplicate )

O O O O O O O B-CONTROLLED_VAR 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

Linearized T22-GFP-H6-FdU dose-response trend line representation compared with unconjugated free oligo-FdU exposure. Antitumor effect was measured as CXCR4+ SW1417 cell viability by MTT after 72 h exposure as the described concentrations. (N=3 experiments in duplicate)


Reduction of cell viability determined by optical microscope images of SW1417 cells exposed to 1µM T22 - GFP - H6 - FdU for 48 h , as compared to T22 - GFP - H6 or free oligo - FdU . ( N = 3 experiments in duplicate ; Scale bar , 100µm ) .

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

Reduction of cell viability determined by optical microscope images of SW1417 cells exposed to 1µM T22-GFP-H6-FdU for 48 h, as compared to T22-GFP-H6 or free oligo-FdU. (N=3 experiments in duplicate; Scale bar, 100µm).


Selective T22 - GFP - H6 - FdU nanoconjugate biodistribution in subcutaneous CXCR4 + SW1417 CRC tumor tissue 5h after a 100µg single intravenous dose , as measured by fluorescence emission using IVIS Spectrum 200 ( N = 5 / group ) . Biodistribution is similar to that achieved by the T22 - GFP - H6 targeting vector and undetectable after Buffer or free oligo - FdU treatment . ( N = 5 mice / group )

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

Selective T22-GFP-H6-FdU nanoconjugate biodistribution in subcutaneous CXCR4+ SW1417 CRC tumor tissue 5h after a 100µg single intravenous dose, as measured by fluorescence emission using IVIS Spectrum 200 (N=5/group). Biodistribution is similar to that achieved by the T22-GFP-H6 targeting vector and undetectable after Buffer or free oligo-FdU treatment. (N=5 mice/group)


Co - localization ( yellow merged ) of the T22 - GFP - H6 - FdU ( green ) and the CXCR4 receptor ( red ) and release of T22 - GFP - H6 - FdU into the cytosol in CXCR4 + tumor cells 5h after a 100ug dose of nanoconjugate , as measured by dual anti - GFP / anti - CXCR4 immunofluorescence ( IF ) . DAPI ( blue nuclear staining ) . Fluorescence emission was measured in the green and red channels using the Image J software and expressed as mean Area ( A ) ± s . e . m ( µm2 ) ( N = 10 , 2 tumor fields x 5 mice ; 200X ) . Note the significant ( P = 0 . 003 ) increase in the area occupied by the green dots ( nanoconjugate realeased to the cell cytosol ) in T22 - GFP - H6 - FdU - treated tumors , compared to free oligo - FdU - treated control tissues . Scale bar , 50 µm .

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

Co-localization (yellow merged) of the T22-GFP-H6-FdU (green) and the CXCR4 receptor (red) and release of T22-GFP-H6-FdU into the cytosol in CXCR4+ tumor cells 5h after a 100ug dose of nanoconjugate, as measured by dual anti-GFP/anti-CXCR4 immunofluorescence (IF). DAPI (blue nuclear staining). Fluorescence emission was measured in the green and red channels using the Image J software and expressed as mean Area (A)±s.e.m (µm2) (N=10, 2 tumor fields x 5 mice; 200X). Note the significant (P=0.003) increase in the area occupied by the green dots (nanoconjugate realeased to the cell cytosol) in T22-GFP-H6-FdU-treated tumors, compared to free oligo-FdU-treated control tissues. Scale bar, 50 µm.


Administration of the CXCR4 antagonist AMD3100 completely blocks T22 - GFP - H6 - FdU tumor biodistribution , as measured by fluorescence emission . Fluorescence is not detected in Buffer or free oligo - FdU controls ( N = 5 tumor fields / group ) .

O O O O O B-CONTROLLED_VAR 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

Administration of the CXCR4 antagonist AMD3100 completely blocks T22-GFP-H6-FdU tumor biodistribution, as measured by fluorescence emission. Fluorescence is not detected in Buffer or free oligo-FdU controls (N=5 tumor fields/group).


The uptake of T22 - GFP - H6 - FdU observed in CXCR4 + SW1417 tumor tissues is almost completely blocked by prior AMD3100 administration , as quantified using the anti - GFP IHC H - score . ( N = 5 tumor fields / group ) .

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 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

The uptake of T22-GFP-H6-FdU observed in CXCR4+ SW1417 tumor tissues is almost completely blocked by prior AMD3100 administration, as quantified using the anti-GFP IHC H-score. (N=5 tumor fields/group).


Representative images of T22 - GFP - H6 - FdU uptake and AMD3100 competition by anti - GFP inmunostaining , which quantitation is reported in panel E . Scale bar , 50

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

Representative images of T22-GFP-H6-FdU uptake and AMD3100 competition by anti-GFP inmunostaining, which quantitation is reported in panel E. Scale bar, 50 µm


Representative images of CXCR4 overexpression in subcutaneous tumor tissue , showing similar CXCR4 levels among compared groups ( N = 5 / group ; Buffer , T22 - GFP - H6 - FdU , T22 - GFP - H6 and free oligo - FdU ) before treatment ( upper panels ) . Representative images of DNA double - strand break induction and caspase 3 activation ( measured with anti - ɣH2AX or anti - cleaved caspase 3 by IHC ) 5h post - administration ( middle panels ) . Apoptotic induction ( Hoescht staining , 24h post - administration , lower panels ) . Note the higher number of cells positive for DSBs , Caspase 3 activation and apoptosis induction in the T22 - GFP - H6 - FdU as compared to free oligo - FdU . Black or white arrows indicate dead cells . Quantitation of the number of cells containing DSBs or active Caspase - 3 in IHC - stained tumor sections 5h post - treatment and the number of condensated or disaggregated nuclei ( by Hoescht staining ) 24h post - treatment in tumor sections of 10 high - powder fields ( 400x magnification ) using the Cell D software . ( N = 50 ; 10 tumor fields / mice ; 5 mice / group ) . Data expressed as mean ± s . e . m .

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

Representative images of CXCR4 overexpression in subcutaneous tumor tissue, showing similar CXCR4 levels among compared groups (N=5/group; Buffer, T22-GFP-H6-FdU, T22-GFP-H6 and free oligo-FdU) before treatment (upper panels). Representative images of DNA double-strand break induction and caspase 3 activation (measured with anti-ɣH2AX or anti-cleaved caspase 3 by IHC) 5h post-administration (middle panels). Apoptotic induction (Hoescht staining, 24h post-administration, lower panels). Note the higher number of cells positive for DSBs, Caspase 3 activation and apoptosis induction in the T22-GFP-H6-FdU as compared to free oligo-FdU. Black or white arrows indicate dead cells. Quantitation of the number of cells containing DSBs or active Caspase-3 in IHC-stained tumor sections 5h post-treatment and the number of condensated or disaggregated nuclei (by Hoescht staining) 24h post-treatment in tumor sections of 10 high-powder fields (400x magnification) using the Cell D software. (N=50; 10 tumor fields/mice; 5 mice/group). Data expressed as mean±s.e.m.


T22 - GFP - H6 - FdU depletes CXCR4 + cancer cells from SW1417 CRC tumor tissue after a 100 µg single dose administration . Note the reduction in CXCR4 + cell fraction in the tumor 24h after injection , their almost complete elimination at 48h and the re - emergence of CXCR4 + cells 72h post - administration , using anti - CXCR4 IHC . In contrast , the CXCR4 + cancer cell fraction ( CXCR4 + CCF ) in tumor tissue remains constant along time after free oligo - FdU treatment . The three day time - lapse for CXCR4 + tumor cell re - appearance defines the dosage interval used in a repeated dose schedule of nanoconjugate administration in the experiments to evaluate its antimetastatic effect . ( N = 5 : 5 mice / group ; 1 samples / mouse ) . Scale bar , 50 µm . Data expressed as mean ± s . e .

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 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

T22-GFP-H6-FdU depletes CXCR4+ cancer cells from SW1417 CRC tumor tissue after a 100 µg single dose administration. Note the reduction in CXCR4+ cell fraction in the tumor 24h after injection, their almost complete elimination at 48h and the re-emergence of CXCR4+ cells 72h post-administration, using anti-CXCR4 IHC. In contrast, the CXCR4+ cancer cell fraction (CXCR4+ CCF) in tumor tissue remains constant along time after free oligo-FdU treatment. The three day time-lapse for CXCR4+ tumor cell re-appearance defines the dosage interval used in a repeated dose schedule of nanoconjugate administration in the experiments to evaluate its antimetastatic effect. (N=5: 5 mice/group; 1 samples/mouse). Scale bar, 50 µm. Data expressed as mean±s.e.m


Significant reduction in the number of spheroid formed ( C . optical microscope ) and their bioluminescence emission ( D , IVIS Spectrum 200 ) , generated by 1x106 disaggregated cells ( cultured in stem cell conditioned media and low - adhesion plates ) , obtained from CXCR4 + luciferase + SW1417 subcutaneous tumors , 24h after 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Buffer - treated or free oligo - FdU - treated mice . D . Quantitation of the bioluminescent signal ( BLI ) expressed as Average Radiant Intensity , obtained using the IVIS spectrum 200 equipment ( N = 2 plates / group ) .

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

Significant reduction in the number of spheroid formed (C. optical microscope) and their bioluminescence emission (D, IVIS Spectrum 200), generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates), obtained from CXCR4+ luciferase+ SW1417 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. D. Quantitation of the bioluminescent signal (BLI) expressed as Average Radiant Intensity, obtained using the IVIS spectrum 200 equipment (N=2 plates/group).


Reduction in the number of formed spheroids ( optical microscope ) generated by 1x106 disaggregated cells ( cultured in stem cell conditioned media and low - adhesion plates ) obtained from CXCR4 + M5 subcutaneous tumors , 24h after 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Buffer - treated or free oligo - FdU - treated mice . ( N = 8 ; 2 mice / group ; 4 plates / mouse ) . ( Scale bar , 100µm ) .

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

Reduction in the number of formed spheroids (optical microscope) generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates) obtained from CXCR4+ M5 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. (N=8; 2 mice/group; 4 plates/mouse). (Scale bar, 100µm).


Reduction in tumor re - initiation capacity after subcutaneous inoculation of 5x106 cells in NSG mice ( N = 4 tumors / group ) derived from disaggregated tumor cells obtained from SC tumors 10 days after administration of 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Free oligo - FdU - treated or Buffer - treated mice . Recording of the number and size of positive tumors . ( N = 4 ; 2 mice / group , 2 injection points / mouse ) .

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

Reduction in tumor re-initiation capacity after subcutaneous inoculation of 5x106 cells in NSG mice (N=4 tumors/group) derived from disaggregated tumor cells obtained from SC tumors 10 days after administration of 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days , as compared to Free oligo-FdU-treated or Buffer-treated mice. Recording of the number and size of positive tumors. (N=4; 2 mice/group, 2 injection points/mouse).


T22 - GFP - H6 - FdU - induced reduction in the number of intravasated tumor emboli in peri - tumoral vessels of the M5 - orthotopic primary tumor ( E . optical images ; F . emboli number quantitation ) and reduction of CXCR4 expression in these emboli ( G ) , treated 7 days after tumor cell implantation with 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Buffer - treated or free oligo - FdU - treated mice . Tumor emboli counting in 10 high powder field at 200x magnification in H & E stained sections from each tumor . ( N = 5 / group ) .

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 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 O O O O O O O O O O

T22-GFP-H6-FdU-induced reduction in the number of intravasated tumor emboli in peri-tumoral vessels of the M5-orthotopic primary tumor (E. optical images; F. emboli number quantitation) and reduction of CXCR4 expression in these emboli (G), treated 7 days after tumor cell implantation with 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. Tumor emboli counting in 10 high powder field at 200x magnification in H&E stained sections from each tumor. (N=5/group).


T22 - GFP - H6 - FdU prevents metastases in the CXCR4 + patient - derived M5 model by potently reducing the total and mean number of liver , lung and peritoneal mets , as recorded in H & E stained histology sections at the end of treatment , in comparison to free oligo - FdU o Buffer - treatment . In contrast , the number of LN mets is not reduced after T22 - GFP - H6 - FdU or free oligo - FdU administration ( N = 6 mice per Buffer group ; N = 7 mice per Free oligo - FdU group and N = 8 mice per T22 - GFP - H6 - FdU group ; 3 samples / mouse ) . Data expressed as mean ± s . e . m .

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

T22-GFP-H6-FdU prevents metastases in the CXCR4+ patient-derived M5 model by potently reducing the total and mean number of liver, lung and peritoneal mets, as recorded in H&E stained histology sections at the end of treatment, in comparison to free oligo-FdU o Buffer-treatment. In contrast, the number of LN mets is not reduced after T22-GFP-H6-FdU or free oligo-FdU administration (N=6 mice per Buffer group; N=7 mice per Free oligo-FdU group and N=8 mice per T22-GFP-H6-FdU group; 3 samples/mouse). Data expressed as mean±s.e.m.


T22 - GFP - H6 - FdU induces a higher reduction of CXCR4 + cancer cell fraction ( CXCR4 + CCF ) in liver , lung and peritoneal metastatic tissue , at the end of treatment , than free oligo - FdU , as measured by anti - CXCR4 IHC . In contrast , T22 - GFP - H6 - FdU or free oligo - FdU does not reduce the CXCR4 + CCF in LN mets or primary tumor tissue after therapy . ( N = 6 mice per Buffer group ; N = 7 mice per Free oligo - FdU group and N = 8 mice per T22 - GFP - H6 - FdU group ; 3 samples / mouse ) . Data expressed as mean ± s . e .

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 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 B-CONTROLLED_VAR 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

T22-GFP-H6-FdU induces a higher reduction of CXCR4+ cancer cell fraction (CXCR4+ CCF) in liver, lung and peritoneal metastatic tissue, at the end of treatment, than free oligo-FdU, as measured by anti-CXCR4 IHC. In contrast, T22-GFP-H6-FdU or free oligo-FdU does not reduce the CXCR4+ CCF in LN mets or primary tumor tissue after therapy. (N=6 mice per Buffer group; N=7 mice per Free oligo-FdU group and N=8 mice per T22-GFP-H6-FdU group; 3 samples/mouse). Data expressed as mean±s.e.m


Representative CXCR4 IHC images of the reduction in CXCR4 + CCF induced by T22 - GFP - H6 - FdU ( or its absence in free oligo - FdU mice ) at the end of treatment , in the M5 patient - derived CRC model , which quantitation is reported in panel B . In the M5 model , the highest reduction in foci number and size occurs in liver metastases , which show the highest reduction in CXCR4 + CCF .

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

Representative CXCR4 IHC images of the reduction in CXCR4+ CCF induced by T22-GFP-H6-FdU (or its absence in free oligo-FdU mice) at the end of treatment, in the M5 patient-derived CRC model, which quantitation is reported in panel B. In the M5 model, the highest reduction in foci number and size occurs in liver metastases, which show the highest reduction in CXCR4+ CCF.


Undetectable T22 - GFP - H6 - FdU emitted fluorescence in normal tissues , except for a transient accumulation 5h after a 100μg dose in the liver , which disappears at 24 h . Liver emitted fluorescence is transient and significantly lower than the one registered in tumor tissue . Tumor / Liver ratio = 7 . 5 ( see tumor intensity in Fig 2B , which was registered in the same experiment ) ( N = 5 mice / group ) . Scale bar , 1 cm .

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

Undetectable T22-GFP-H6-FdU emitted fluorescence in normal tissues, except for a transient accumulation 5h after a 100μg dose in the liver, which disappears at 24 h. Liver emitted fluorescence is transient and significantly lower than the one registered in tumor tissue. Tumor/Liver ratio= 7.5 (see tumor intensity in Fig 2B, which was registered in the same experiment) (N=5 mice/group). Scale bar, 1 cm.


Representative images depicting the level of DNA double strand break ( DSB ) induction in histologically normal bone marrow 5h after treatment , as measured by anti - ɣ - H2AX , which is higher in free oligo - FdU - treated mice than in T22 - GFP - H6 - FdU ( P = 0 . 047 ) . Low level of cells containing DSBs in histologically normal kidney after T22 - GFP - H6 - FdU or free oligo - FdU treatment , a finding occurring in all normal tissues analysed . ( N = 50 , 5 mice / group ; 10 fields / mouse ) .

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

Representative images depicting the level of DNA double strand break (DSB) induction in histologically normal bone marrow 5h after treatment, as measured by anti-ɣ-H2AX, which is higher in free oligo-FdU-treated mice than in T22-GFP-H6-FdU (P=0.047). Low level of cells containing DSBs in histologically normal kidney after T22-GFP-H6-FdU or free oligo-FdU treatment, a finding occurring in all normal tissues analysed. (N=50, 5 mice/group; 10 fields/mouse).


Representative images showing lack of histopathological alterations in H & E stained tissue or apoptotic induction in H & E stained samples of CXCR4 + ( bone marrow ) and CXCR4 - ( brain , kidney , liver , lung and heart ) normal tissues 24h after the administration of a 100 μg dose of T22 - GFP - H6 - FdU or an equimolecular dose of free oligo - FdU ( N = 5 / group ) . Note that the transient nanoconjugate distribution to liver or the DNA damage induced in bone marrow do not lead to cytoxicity on these no - tumor tissues . ( N = 50 , 5 mice / group ; 10 fields / mouse )

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

Representative images showing lack of histopathological alterations in H&E stained tissue or apoptotic induction in H&E stained samples of CXCR4+ (bone marrow) and CXCR4- (brain, kidney, liver, lung and heart) normal tissues 24h after the administration of a 100 μg dose of T22-GFP-H6-FdU or an equimolecular dose of free oligo-FdU (N=5/group). Note that the transient nanoconjugate distribution to liver or the DNA damage induced in bone marrow do not lead to cytoxicity on these no-tumor tissues. (N=50, 5 mice/group; 10 fields/mouse)


Lack of differences in body weight among groups registered along time in the SW1417 - derived CCR model and the regression of metastases protocol ( N = 10 mice / group ) . Lack of differences in body mouse weight among groups registered along time in the SW1417 cell line - derived model and the prevention of metastasis protocol ( Buffer ( N = 11 ; Free oligo - FdU ( N = 12 ) , T22 - GFP - H6 - FdU ( N = 12 ) ) . Lack of differences in body mouse weight among groups registered along time in the M5 patient - derived model and the prevention of metastasis protocol ( Buffer ( N = 6 ) ; Free oligo - FdU ( N = 17 ) ; T22 - GFP - H6 - FdU ( N = 8 ) . Scale bar , 100

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

Lack of differences in body weight among groups registered along time in the SW1417-derived CCR model and the regression of metastases protocol (N=10 mice/group). Lack of differences in body mouse weight among groups registered along time in the SW1417 cell line-derived model and the prevention of metastasis protocol (Buffer (N=11; Free oligo-FdU (N=12), T22-GFP-H6-FdU (N=12)). Lack of differences in body mouse weight among groups registered along time in the M5 patient-derived model and the prevention of metastasis protocol (Buffer (N=6); Free oligo-FdU (N=17); T22-GFP-H6-FdU (N=8). Scale bar, 100 μm


Serum cytokine levels from control - treated ( black ) and anti - HER2 / CD3 TDB - treated ( red ) mice , 6 h and 24 h post treatment ( n = 9 mice / treatment ) .

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

Serum cytokine levels from control-treated (black) and anti-HER2/CD3 TDB-treated (red) mice, 6 h and 24 h post treatment (n = 9 mice/treatment).


G , H . Representative images of tumor ( G ) or liver ( H ) sections from the Founder5 model stained with CD3 ( red ) , Endomucin ( white ) , and DAPI ( blue ) . Right panels : quantification of CD3 + cell counts in Founder5 whole tumor ( G ) or liver ( H ) sections normalized to tissue areas . All samples were analyzed 24 h post treatment , n = 8 / 9 mice / treatment .

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

G, H. Representative images of tumor (G) or liver (H) sections from the Founder5 model stained with CD3 (red), Endomucin (white), and DAPI (blue). Right panels: quantification of CD3+ cell counts in Founder5 whole tumor (G) or liver (H) sections normalized to tissue areas. All samples were analyzed 24 h post treatment, n = 8/9 mice/treatment.


Tumor ( I ) tissue cytokine levels normalized to total protein concentration from control - treated ( black ) and anti - HER2 / CD3 TDB - treated ( red ) mice , 24 h post treatment ( n = 8 mice / treatment ) .

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

Tumor (I) tissue cytokine levels normalized to total protein concentration from control-treated (black) and anti-HER2/CD3 TDB-treated (red) mice, 24 h post treatment (n = 8 mice/treatment).


B , C . Flow cytometry analysis of T cell phenotype and T cell subsets for CD8 + T cells ( B ) and CD4 + T cells ( C ) in dissociated tumors normalized to tumor weights ( upper panels ) or dissociated livers normalized to liver weights ( lower panels ) , 24 h post treatment . ( n = 8 mice / treatment ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Flow cytometry analysis of T cell phenotype and T cell subsets for CD8+ T cells (B) and CD4+ T cells (C) in dissociated tumors normalized to tumor weights (upper panels) or dissociated livers normalized to liver weights (lower panels), 24 h post treatment. (n = 8 mice/treatment).


Cytokine concentrations in conditioned medium of HUVECs cultured without treatment ( blue diamond , treatment b ) , or with 600 pg / ml a ( pink circle , treatment c ) , or with control PBMC supernatant ( orange triangle , treatment d ) , or with TDB - conditioned PBMC supernatant ( purple square , treatment e ) . All done in n = 6 replicates with 6 hours incubation . Endothelial cell culture medium alone ( black circle , treatment a , n = 4 ) was plotted as reference .

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

Cytokine concentrations in conditioned medium of HUVECs cultured without treatment (blue diamond, treatment b), or with 600 pg/ml a (pink circle, treatment c), or with control PBMC supernatant (orange triangle, treatment d), or with TDB-conditioned PBMC supernatant (purple square, treatment e). All done in n = 6 replicates with 6 hours incubation. Endothelial cell culture medium alone (black circle, treatment a, n = 4) was plotted as reference.


Gating strategy for flow cytometry analysis of surface expression of ICAM - 1 and VCAM - 1 on HUVECs . Singlets were gated based on forward scatter area ( FSC - A ) versus forward scatter height ( FSC - H ) . Activated endothelial cells were gated as ICAM - 1 + or VCAM - 1 + endothelial cells from viable cells . Quantification of percentage of ICAM - 1 + ( top panel ) and VCAM - 1 + ( bottom panel ) endothelial cells with the same treatments as defined in panel B . All done in triplicates .

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

Gating strategy for flow cytometry analysis of surface expression of ICAM-1 and VCAM-1 on HUVECs. Singlets were gated based on forward scatter area (FSC-A) versus forward scatter height (FSC-H). Activated endothelial cells were gated as ICAM-1+ or VCAM-1+ endothelial cells from viable cells.   Quantification of percentage of ICAM-1+ (top panel) and VCAM-1+ (bottom panel) endothelial cells with the same treatments as defined in panel B. All done in triplicates.


A , B . Representative images of liver ( A ) or tumor ( B ) sections from the Founder5 tumor - bearing mice stained with ICAM ‑ 1 ( red ) , VCAM - 1 ( white ) , and Endomucin ( green ) 24 hours post treatment . Right panels are enlarged views of the boxed areas showing only ICAM - 1 and VCAM - 1 . Scale bar = 100 μm . Below : quantification of total ICAM - 1 + and VCAM - 1 + areas as well as vascular ( vasc . ) ICAM ‑ 1 + ( ICAM - 1 + Endomucin + ) and vascular VCAM - 1 + ( VCAM ‑ 1 + Endomucin + ) areas normalized to tissue areas . n = 9 mice / treatment .

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

A, B. Representative images of liver (A) or tumor (B) sections from the Founder5 tumor-bearing mice stained with ICAM‑1 (red), VCAM-1 (white), and Endomucin (green) 24 hours post treatment. Right panels are enlarged views of the boxed areas showing only ICAM-1 and VCAM-1. Scale bar = 100 μm. Below: quantification of total ICAM-1+ and VCAM-1+ areas as well as vascular (vasc.) ICAM‑1+ (ICAM-1+ Endomucin+) and vascular VCAM-1+ (VCAM‑1+ Endomucin+) areas normalized to tissue areas. n = 9 mice/treatment.


E , F . Densities of total blood vessels ( left panel ) and mature blood vessels ( right panel ) in liver ( E ) and tumor ( F ) sections from the Founder5 tumor - bearing mice normalized to tissue areas . n = 9 mice / treatment .

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

E, F. Densities of total blood vessels (left panel) and mature blood vessels (right panel) in liver (E) and tumor (F) sections from the Founder5 tumor-bearing mice normalized to tissue areas. n = 9 mice/treatment.