( A - G ) Lateral ( A - B ) , ventrolateral ( C - F ) and ventral ( G ) images of the cranial vessels in an etv2 : EGFP ; kdrl : nlsmCherry compound transgenic embryo , which has had the etv2 - expressing angioblast population ( early VA - L ) on the left lateral side of the yolk sac ablated . Images were taken at 30 hpf before ( A ) and after ( B ) ablation of the early VA - L , with the site of ablation indicated ( B ; blue dotted circle ) . Note that the LDA and VA adjacent to the VA - L become photobleached during the ablation process ( B ) , however these were not ablated as they can be seen developing normally at 54 hpf ( D ) . Follow - up images were taken of both the contralateral ( C , E ) and ablated ( D , F ) sides of the embryo ' s head at 54 hpf ( C - D ) and 78 hpf ( E - G ) . Although the VA - L ( light blue dotted line ) is seen migrating towards the FLS ( white dotted lined ) on the contralateral side at 54 hpf ( C ) , it is missing from the ablated side ( D ) . Subsequently , the LFL ( white dotted line ) of the ablated side is short ( F ) compared to that of the contralateral side ( E ) . In addition , the HA ( pink dotted line ) is underdeveloped on the ablated side compared to the contralateral side , as it no longer extends towards nor connects to the left AA1 ( G ) . ( H ) Quantitation of the length of the LFL ( µm ) on the contralateral ( control ) side and on the ablated side at 78 hpf ( n = 16 ) . ( I ) Quantitation of the total number of cells within the LFL on the contralateral ( control ) side and from the ablated side at 78 hpf ( n = 16 ) .

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

(A-G) Lateral (A-B), ventrolateral (C-F) and ventral (G) images of the cranial vessels in an etv2:EGFP;kdrl:nlsmCherry compound transgenic embryo, which has had the etv2-expressing angioblast population (early VA-L) on the left lateral side of the yolk sac ablated. Images were taken at 30 hpf before (A) and after (B) ablation of the early VA-L, with the site of ablation indicated (B; blue dotted circle). Note that the LDA and VA adjacent to the VA-L become photobleached during the ablation process (B), however these were not ablated as they can be seen developing normally at 54 hpf (D). Follow-up images were taken of both the contralateral (C, E) and ablated (D, F) sides of the embryo's head at 54 hpf (C-D) and 78 hpf (E-G). Although the VA-L (light blue dotted line) is seen migrating towards the FLS (white dotted lined) on the contralateral side at 54 hpf (C), it is missing from the ablated side (D). Subsequently, the LFL (white dotted line) of the ablated side is short (F) compared to that of the contralateral side (E). In addition, the HA (pink dotted line) is underdeveloped on the ablated side compared to the contralateral side, as it no longer extends towards nor connects to the left AA1 (G). (H) Quantitation of the length of the LFL (µm) on the contralateral (control) side and on the ablated side at 78 hpf (n = 16). (I) Quantitation of the total number of cells within the LFL on the contralateral (control) side and from the ablated side at 78 hpf (n = 16).


a , Flow chart of an in vivo screen to identify active DUBs by using biotin - Ub - VME pull - down assay ( left panel ) , in which the USP8 peptide was identified by mass - spectrometry analysis ( right panel ) . Silver staining and immunoblot ( middle panel ) of precipitants pulled down by biotin - Ub - VME probe of MDA - MB - 231 cells .

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

a, Flow chart of an in vivo screen to identify active DUBs by using biotin-Ub-VME pull-down assay (left panel), in which the USP8 peptide was identified by mass-spectrometry analysis (right panel). Silver staining and immunoblot (middle panel) of precipitants pulled down by biotin-Ub-VME probe of MDA-MB-231 cells.


b , Experimental procedure in vivo : flow chart and pictures of an in vivo screen to identify DUBs that promote breast cancer metastasis in mice ( left panel ) . Low metastatic MDA - MB - 231 - Luciferase / GFP breast cancer cells were infected with viral vectors expressing DUB cDNAs and subsequently intracardially injected into nude mice . The mice were monitored for 4 weeks by in vivo BLI : of the 30 mice injected with cells expressing DUB cDNA pool ; 3 had metastases in the brain and bone ( 3 / 30 ) , 4 had metastases on the back and in the bone ( 4 / 30 ) , and 9 had micro - metastasis ( 9 / 30 ) ; the 30 mice injected with control cells showed no detectable metastasis ( 30 / 30 ) . Right panel : hints of the identified DUBs were shown .

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b, Experimental procedure in vivo: flow chart and pictures of an in vivo screen to identify DUBs that promote breast cancer metastasis in mice (left panel). Low metastatic MDA-MB-231-Luciferase/GFP breast cancer cells were infected with viral vectors expressing DUB cDNAs and subsequently intracardially injected into nude mice. The mice were monitored for 4 weeks by in vivo BLI: of the 30 mice injected with cells expressing DUB cDNA pool; 3 had metastases in the brain and bone (3/30), 4 had metastases on the back and in the bone (4/30), and 9 had micro-metastasis (9/30); the 30 mice injected with control cells showed no detectable metastasis (30/30). Right panel: hints of the identified DUBs were shown.


c , The expression distribution of USP8 gene in breast cancer tumor tissues ( n = 1097 ) and normal tissues ( n = 572 ) , where the horizontal axis represents different groups of samples , the vertical axis represents the gene expression distribution ( left panel ) . The box - and - whisker plots represent the medians ( middle line ) , first quartiles ( lower bound line ) , third quartiles ( upper bound line ) , and the ± 1 . 5 × interquartile ranges ( whisker lines ) . Kaplan - Meier curves showing the metastasis - free survival of individuals was negatively correlated with USP8 expression by log - rank test ( right panel ) .

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c, The expression distribution of USP8 gene in breast cancer tumor tissues (n = 1097) and normal tissues (n = 572), where the horizontal axis represents different groups of samples, the vertical axis represents the gene expression distribution (left panel). The box-and-whisker plots represent the medians (middle line), first quartiles (lower bound line), third quartiles (upper bound line), and the ±1.5× interquartile ranges (whisker lines). Kaplan-Meier curves showing the metastasis-free survival of individuals was negatively correlated with USP8 expression by log-rank test (right panel).


d , MCF10A - RAS cells infected with control ( Co . sh ) or USP8 shRNA lentivirus ( # 1 and # 2 ) were analyzed in a tumor sphere assay . The number of the mammospheres with diameter > 60 μm was quantified . The graph shows the number ( No . ) of tumor spheres per 5 × 103 cells seeded . Scale bar 50

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d, MCF10A-RAS cells infected with control (Co.sh) or USP8 shRNA lentivirus (# 1 and # 2) were analyzed in a tumor sphere assay. The number of the mammospheres with diameter > 60 μm was quantified. The graph shows the number (No.) of tumor spheres per 5 × 103 cells seeded. Scale bar 50 μm


e , MCF10A - RAS cells infected with control ( Co . sh ) or USP8 shRNA lentivirus ( # 1 and # 2 ) were subcutaneously injected into nude mice at the indicated numbers . Mean of tumor volumes at week 5 ( n = 10 mice for each group ) .

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e, MCF10A-RAS cells infected with control (Co.sh) or USP8 shRNA lentivirus (# 1 and # 2) were subcutaneously injected into nude mice at the indicated numbers. Mean of tumor volumes at week 5 (n = 10 mice for each group).


f , Microscopy pictures of representative images of zebrafish tail fin injected with control or USP8 stably depleted - MDA - MB - 231 cells at 5 days post injection , n = 20 for each group . g , The final invasive areas of zebrafish embryos displaying invasion and experimental metastasis at 5 dpi ( left panel ) ; Percentage of embryos injected with control or USP8 stably depleted MDA - MB - 231 cells ( # 1 and # 2 ) displaying metastasis at 1 , 3 and 5 days post injection ( right panel ) .

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f, Microscopy pictures of representative images of zebrafish tail fin injected with control or USP8 stably depleted-MDA-MB-231 cells at 5 days post injection, n = 20 for each group. g, The final invasive areas of zebrafish embryos displaying invasion and experimental metastasis at 5 dpi (left panel); Percentage of embryos injected with control or USP8 stably depleted MDA-MB-231 cells (# 1 and # 2) displaying metastasis at 1, 3 and 5 days post injection (right panel).


h , Bioluminescent images of three representative mice from each group ( n = 6 ) at week 6 injected into left heart ventricle with control or MDA - MB - 231 cells stably depleted of endogenous USP8 with two independent shRNA ( # 1 and # 2 ) . i , BLI signal ( left panel ) , number of bone metastasis of every mouse ( middle panel ) and the percentage of bone metastasis free mice in each experimental group ( n = 6 mice per group ) followed in time as in h ( right panel ) .

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h, Bioluminescent images of three representative mice from each group (n = 6) at week 6 injected into left heart ventricle with control or MDA-MB-231 cells stably depleted of endogenous USP8 with two independent shRNA (# 1 and # 2). i, BLI signal (left panel), number of bone metastasis of every mouse (middle panel) and the percentage of bone metastasis free mice in each experimental group (n = 6 mice per group) followed in time as in h (right panel).


a , Heatmap of USP8 - correlated genes in a comparison of breast cancer patients with USP8 - high ( n = 38 ) versus with USP8 - low ( n = 39 ) .

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a, Heatmap of USP8-correlated genes in a comparison of breast cancer patients with USP8-high (n = 38) versus with USP8-low (n = 39).


b , Pre - ranked gene set enrichment analysis ( GSEA ) in USP8 - high ( n = 38 ) versus USP8 - low ( n = 39 ) breast cancer patients .

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b, Pre-ranked gene set enrichment analysis (GSEA) in USP8-high (n = 38) versus USP8-low (n = 39) breast cancer patients.


c , Experimental procedure in vivo : MDA - MB - 231 cells were intracardially injected into nude mice ( n = 10 ) . qRT - PCR analysis of bone metastatic cells showing the correlations between USP8 and Snai1 or Snai2 ( Slug ) in cells isolated from mutiple metastatic nodules ( n = 21 ) from mice .

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c, Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice (n = 10). qRT-PCR analysis of bone metastatic cells showing the correlations between USP8 and Snai1 or Snai2 (Slug) in cells isolated from mutiple metastatic nodules (n = 21) from mice.


d , 3D spheroid invasion assay of MCF10A - Ras cells showing USP8 wt increased TGF - β - induced invasion . Relative invasion was quantified and shown in the right panel .

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d, 3D spheroid invasion assay of MCF10A-Ras cells showing USP8 wt increased TGF-β-induced invasion. Relative invasion was quantified and shown in the right panel.


e , MCF10A - Ras cells stably expressing control vector ( Co . vec ) , USP8 wt or USP8 cs were injected into the blood circulation of 48 - hpf zebrafish embryos . SB431542 ( 5 μM ) was added to the zebrafish environment . Representative images of zebrafish at 5 dpi ( left panel ) ; the experimental metastatic areas ( middle panel ) and percentage of embryos showing metastasis ( right panel ) are shown , n = 20 .

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e, MCF10A-Ras cells stably expressing control vector (Co.vec), USP8 wt or USP8 cs were injected into the blood circulation of 48-hpf zebrafish embryos. SB431542 (5 μM) was added to the zebrafish environment. Representative images of zebrafish at 5 dpi (left panel); the experimental metastatic areas (middle panel) and percentage of embryos showing metastasis (right panel) are shown, n = 20.


f , Diagram of DUB cDNA screening data of TGF - β - induced SMAD3 / SMAD4 - dependent CAGA12 - Luc transcriptional reporter in HEK293T cells . The X - and Y - axes are the relative luciferase activity in two replicates .

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f, Diagram of DUB cDNA screening data of TGF-β-induced SMAD3/ SMAD4-dependent CAGA12-Luc transcriptional reporter in HEK293T cells. The X- and Y-axes are the relative luciferase activity in two replicates.


g , CAGA12 - Luc transcriptional response of HEK293T cells transfected with USP8 wt / cs or shUSP8 ( # 1 and # 2 ) as indicated and treated with TGF - β ( 1 ng / ml ) overnight .

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g, CAGA12-Luc transcriptional response of HEK293T cells transfected with USP8 wt/cs or shUSP8 (#1 and #2) as indicated and treated with TGF-β (1 ng/ml) overnight.


h , Heatmap of TGF - β target genes by qRT - PCR analysis in control or MCF10A - RAS cells stably depleting USP8 ( # 1 and # 2 shRNA ) or expressing USP8 wt / cs and treated with or without TGF - β ( 2 . 5 ng / ml ) for 8 hrs . Appendix Table S2 for details .

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

h, Heatmap of TGF-β target genes by qRT-PCR analysis in control or MCF10A-RAS cells stably depleting USP8 (#1 and #2 shRNA) or expressing USP8 wt/cs and treated with or without TGF-β (2.5 ng/ml) for 8 hrs. Appendix Table S2 for details.


i , Immunoblot analysis of total cell lysates , immunoprecipitates derived from control and USP8 stably depleted ( # 1 and # 2 shRNA ) ( left panel ) and USP8 wt / cs over - expressed ( right panel ) MDA - MB - 231 cells treated with TGF - β ( 2 . 5 ng / ml ) for indicated time points .

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i, Immunoblot analysis of total cell lysates, immunoprecipitates derived from control and USP8 stably depleted (#1 and #2 shRNA) (left panel) and USP8 wt/cs over-expressed (right panel) MDA-MB-231 cells treated with TGF-β (2.5 ng/ml) for indicated time points.


j , Immunofluorescence and 4 , 6 - diamidino - 2 - phenylindole ( DAPI ) staining of HaCaT cells infected with control or USP8 wt / cs and treated with TGF - β ( 2 . 5 ng / ml ) and SB431542 ( 10 μM ) for 72 hrs . Scale bar , 20 μm .

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j, Immunofluorescence and 4, 6-diamidino-2-phenylindole (DAPI) staining of HaCaT cells infected with control or USP8 wt/cs and treated with TGF-β (2.5 ng/ml) and SB431542 (10 μM) for 72 hrs. Scale bar, 20 μm.


k , Immunoblot analysis of cell lysate of control and USP8 stably depleted - MCF10AR - RAS ( MII ) cells treated with TGF - β ( 5 ng / ml ) for 48 hrs ( left ) ; Immunoblot analysis of cell lysate of control and USP8 wt or USP8 cs stably expressed - MCF10AR - RAS ( MII ) cells treated with TGF - β ( 5 ng / ml ) or SB431542 ( 10 μM ) for 48 hrs ( right ) .

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k, Immunoblot analysis of cell lysate of control and USP8 stably depleted-MCF10AR-RAS (MII) cells treated with TGF-β (5 ng/ml) for 48 hrs (left); Immunoblot analysis of cell lysate of control and USP8 wt or USP8 cs stably expressed-MCF10AR-RAS (MII) cells treated with TGF-β (5 ng/ml) or SB431542 (10 μM) for 48 hrs (right).


a , Immunofluorescence and DAPI staining of HeLa cells transfected with TβRII - HA and Flag - USP8 . Scale bar , 10 μm .

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a, Immunofluorescence and DAPI staining of HeLa cells transfected with TβRII-HA and Flag-USP8. Scale bar, 10 μm.


b , USP8 interacts with TβRII . Purified prokaryotically expressed USP8 and recombinant GST - TβRII - ICD were incubated in vitro and immunoprecipitated with anti - USP8 antibody ; immunoprecipitates were then immunoblotted for TβRII - ICD and USP8 ( top panel ) ; Immunoblot analysis of total cell lysate derived from MDA - MB - 231 cells treated with TGF - β as indicated and immunoprecipitated with control IgG ( ns ) or anti - TβRII antibody ( bottom panel ) .

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b, USP8 interacts with TβRII. Purified prokaryotically expressed USP8 and recombinant GST-TβRII-ICD were incubated in vitro and immunoprecipitated with anti-USP8 antibody; immunoprecipitates were then immunoblotted for TβRII-ICD and USP8 (top panel); Immunoblot analysis of total cell lysate derived from MDA-MB-231 cells treated with TGF-β as indicated and immunoprecipitated with control IgG (ns) or anti-TβRII antibody (bottom panel).


c , Immunoblot analysis of anti - TβRII immunoprecipitates from HEK293T cells stably expressing HA - Ub and in vitro incubated with purified USP8 wt / cs .

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c, Immunoblot analysis of anti-TβRII immunoprecipitates from HEK293T cells stably expressing HA-Ub and in vitro incubated with purified USP8 wt/cs.


d , IB of Nickle - pull down precipitants derived from His - Ub stably expressed HEK293T cells incubated with purified USP8 protein as indicated time points .

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d, IB of Nickle-pull down precipitants derived from His-Ub stably expressed HEK293T cells incubated with purified USP8 protein as indicated time points.


e , IB of TCL and immunoprecipitants derived from HEK293T cells transfected with indicated plasmids and treat with or without MG132 ( 10 μM ) for 4 h .

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e, IB of TCL and immunoprecipitants derived from HEK293T cells transfected with indicated plasmids and treat with or without MG132 (10 μM) for 4 h.


f , IB of TCL and immunoprecipitants derived from HA - Ub stably expressed HEK293T cells transfected with Myc - USP8 wt / cs ( left panel ) or infected with control ( Co . sh ) or USP8 shRNA lentivirus ( # 1 and # 2 ) ( right panel ) and treated with TGF - β ( 5 ng / ml ) as indicated .

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f, IB of TCL and immunoprecipitants derived from HA-Ub stably expressed HEK293T cells transfected with Myc-USP8 wt/cs (left panel) or infected with control (Co.sh) or USP8 shRNA lentivirus (#1 and #2) (right panel) and treated with TGF-β (5 ng/ml) as indicated.


g , IB of HeLa cells stably expressing TβRII - HA and transfected with control empty vector ( Co . vector ) and Flag - USP8 wt / cs plasmids and treated with CHX ( 20 μg / ml ) for indicated time points .

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g, IB of HeLa cells stably expressing TβRII-HA and transfected with control empty vector (Co.vector) and Flag-USP8 wt/cs plasmids and treated with CHX (20 μg/ml) for indicated time points.


h , Immunoblot analysis of MCF10A - RAS cells stably expressing USP8 wt / cs or USP8 shRNA ( # 1 and # 2 ) .

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h, Immunoblot analysis of MCF10A-RAS cells stably expressing USP8 wt/cs or USP8 shRNA (#1 and #2).


i , Immunoblot analysis of biotinylated TβRII in MDA - MB - 231 cells ectopically expressing USP8 wt / cs and treated with TGF - β ( 5 ng / ml ) as indicated ( left panel ) . Quantification of the band intensities is shown in right panel . Band intensity was normalized to the t = 0 controls ( right panel ) . n = 2 technical replicates .

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i, Immunoblot analysis of biotinylated TβRII in MDA-MB-231 cells ectopically expressing USP8 wt/cs and treated with TGF-β (5 ng/ml) as indicated (left panel). Quantification of the band intensities is shown in right panel. Band intensity was normalized to the t=0 controls (right panel). n = 2 technical replicates.


j , Immunoblot analysis of biotinylated TβRII in MDA - MB - 231 cells infected with lentivirus encoding control shRNA ( Co . sh ) , USP8 shRNA ( # 1 and # 2 ) and treated with TGF - β ( 5 ng / ml ) as indicated ( left panel ) . Quantification of the band intensities is shown in right panel . Band intensity was normalized to the t = 0 controls ( right panel ) .

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j, Immunoblot analysis of biotinylated TβRII in MDA-MB-231 cells infected with lentivirus encoding control shRNA (Co.sh), USP8 shRNA (# 1 and # 2) and treated with TGF-β (5 ng/ml) as indicated (left panel). Quantification of the band intensities is shown in right panel. Band intensity was normalized to the t=0 controls (right panel).


l , Expression of USP8 of normal tissues ( n = 11 ) and breast cancer patients ( n = 110 ) .

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l, Expression of USP8 of normal tissues (n = 11) and breast cancer patients (n = 110).


m , Scatter - plot showing the positive correlation of USP8 , TβRII and p - SMAD2 expression in patients ( n = 110 ) . H score of every tissue samples of USP8 , TβRII and p - SMAD2 was calculated .

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m, Scatter-plot showing the positive correlation of USP8, TβRII and p-SMAD2 expression in patients (n = 110). H score of every tissue samples of USP8, TβRII and p-SMAD2 was calculated.


n , Percentage of specimens displaying low or high USP8 expression compared to the expression levels of TβRII and P - SMAD2 in human breast cancer tissue ( n = 110 ) . p values from Student ' s t tests were indicated .

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n, Percentage of specimens displaying low or high USP8 expression compared to the expression levels of TβRII and P-SMAD2 in human breast cancer tissue (n = 110). p values from Student's t tests were indicated.


o , GEPIA analysis curves showing the metastasis - free survival of breast cancer patients was significantly correlated with USP8 expression by log - rank test in the TCGA and GTEx datasets ( http : / / gepia . cancer - pku . cn / ) . Cutoff - high = 67 % , cutoff - low = 25 % .

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o, GEPIA analysis curves showing the metastasis-free survival of breast cancer patients was significantly correlated with USP8 expression by log-rank test in the TCGA and GTEx datasets (http://gepia.cancer-pku.cn/). Cutoff-high = 67%, cutoff-low = 25%.


a , Comparison of the pre - treatment levels of USP8 mRNA in patient - derived tissue samples by qRT - PCR ( left panel ) , enzyme activity of USP8 in patient - derived tissue samples by Ub - VME ( middle panel ) and TβRII + crEVs level in the plasma from breast cancer patients by ELISA ( right panel ) between breast cancer patients with or without clinical response to PTX . R , responders ; n = 16 ; NR , non - responders ; n = 14 .

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a, Comparison of the pre-treatment levels of USP8 mRNA in patient-derived tissue samples by qRT-PCR (left panel), enzyme activity of USP8 in patient-derived tissue samples by Ub-VME (middle panel) and TβRII+ crEVs level in the plasma from breast cancer patients by ELISA (right panel) between breast cancer patients with or without clinical response to PTX. R, responders; n = 16; NR, non-responders; n = 14.


b , Pearson correlation of the USP8 mRNA or USP8 enzyme activity in patient - derived tissue samples with the EV - TβRII levels in the plasma from breast cancer patients without clinical response to PTX as in a ( NR , n = 14 ) .

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b, Pearson correlation of the USP8 mRNA or USP8 enzyme activity in patient-derived tissue samples with the EV-TβRII levels in the plasma from breast cancer patients without clinical response to PTX as in a (NR, n = 14).


c , Objective response rate ( ORR ) for patients as in a ( n = 30 ) with high ( > 0 . 95 ng / ml ) and low ( < 0 . 95 ng / ml ) pre - treatment levels of circulating EV - TβRII .

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c, Objective response rate (ORR) for patients as in a (n = 30) with high (>0.95 ng/ml) and low (<0.95 ng/ml) pre-treatment levels of circulating EV-TβRII.


d , Pearson correlation of the percentage of IFN - γ + of CD8 + cells to the TβRII + crEV level in the plasma from breast cancer patients without clinical response to PTX as in a ( NR , n = 14 ) .

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d, Pearson correlation of the percentage of IFN-γ+ of CD8+ cells to the TβRII+ crEV level in the plasma from breast cancer patients without clinical response to PTX as in a (NR, n = 14).


e , TβRII + crEVs ( left panel ) , the USP8 enzyme activity ( midde panel ) and USP8 mRNA ( right panel ) from breast cancer patients with or without clinical response to combined therapy using anti - PD - L1 ( Atezolizumab ) and paclitaxel . R , responders ; n = 10 ( left panel ) , n = 5 ( middle and right panels , only 5 patient - derived tissue samples obtained ) . NR , non - responders ; n = 10 ( left panel ) , n = 6 ( middle and right panels , only 5 patient - derived tissue samples obtained ) .

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e, TβRII+ crEVs (left panel), the USP8 enzyme activity (midde panel) and USP8 mRNA (right panel) from breast cancer patients with or without clinical response to combined therapy using anti-PD-L1 (Atezolizumab) and paclitaxel. R, responders; n = 10 (left panel), n = 5 (middle and right panels, only 5 patient-derived tissue samples obtained). NR, non-responders; n = 10 (left panel), n = 6 (middle and right panels, only 5 patient-derived tissue samples obtained).


Experimental procedure in vivo : MDA - MB - 231 cells were intracardially injected into nude mice . Bioluminescent imaging ( BLI ) of representative mice from each group at week 6 injected with control ( n = 9 ) or MDA - MB - 231 cells stably ovexpressed with USP8 - WT ( n = 7 ) or USP8 - CS ( n = 9 ) . BLI images are shown ( f ( left panel ) ) . BLI signal of each mice ( f ( middle panel ) ) and number of bone metastasis ( f ( right panel ) ) of every

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

Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. Bioluminescent imaging (BLI) of representative mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). BLI images are shown (f (left panel)). BLI signal of each mice (f (middle panel)) and number of bone metastasis (f (right panel)) of every mouse


Experimental procedure in vivo : MDA - MB - 231 cells were intracardially injected into nude mice . mice from each group at week 6 injected with control ( n = 9 ) or MDA - MB - 231 cells stably ovexpressed with USP8 - WT ( n = 7 ) or USP8 - CS ( n = 9 ) . the percentage of bone metastasis free mice survival in each experimental group followed in time ( g ) .

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Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). the percentage of bone metastasis free mice survival in each experimental group followed in time (g).


Experimental procedure in vivo : MDA - MB - 231 cells were intracardially injected into nude mice . mice from each group at week 6 injected with control ( n = 9 ) or MDA - MB - 231 cells stably ovexpressed with USP8 - WT ( n = 7 ) or USP8 - CS ( n = 9 ) . Fold change of circulating EV - TβRII by ELISA analysis in plasma samples of mice in each experimental group at week 6 ( h ) .

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

Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice. mice from each group at week 6 injected with control (n = 9) or MDA-MB-231 cells stably ovexpressed with USP8-WT (n = 7) or USP8-CS (n = 9). Fold change of circulating EV-TβRII by ELISA analysis in plasma samples of mice in each experimental group at week 6 (h).


nude mice were tail vein - injected with 4T07 - Luc cells ( 5 × 105 cells per mouse ) expressing control vector or doxycycline - inducible USP8 - wt / cs and tumors were grown for 2 weeks , followed by the administration of doxycycline ( n = 6 for each group ) Normalized BLI signals ( left panel ) and representative mice ( right panel ) from each group at the indicated times ( j ) .

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nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) Normalized BLI signals (left panel) and representative mice (right panel) from each group at the indicated times (j).


nude mice were tail vein - injected with 4T07 - Luc cells ( 5 × 105 cells per mouse ) expressing control vector or doxycycline - inducible USP8 - wt / cs and tumors were grown for 2 weeks , followed by the administration of doxycycline ( n = 6 for each group ) Immunoblot analysis of the circulating EVs from plasma in mice ( k ) .

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nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) Immunoblot analysis of the circulating EVs from plasma in mice (k).


nude mice were tail vein - injected with 4T07 - Luc cells ( 5 × 105 cells per mouse ) expressing control vector or doxycycline - inducible USP8 - wt / cs and tumors were grown for 2 weeks , followed by the administration of doxycycline ( n = 6 for each group ) ELISA analysis of circulating exosomal TβRII level in plasma samples of mice at the indicated times ( l ) .

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nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) ELISA analysis of circulating exosomal TβRII level in plasma samples of mice at the indicated times (l).


a , Gene signatures indicating the Golgi - associated vesicle biogenesis - and membrane trafficking - related genes are significantly enriched in USP8 - high ( n = 38 ) patients versus USP8 - low ( n = 39 ) expressing patients ; shown by pre - ranked gene - set enrichment analysis ( GSEA ) .

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a, Gene signatures indicating the Golgi-associated vesicle biogenesis- and membrane trafficking-related genes are significantly enriched in USP8-high (n = 38) patients versus USP8-low (n = 39) expressing patients; shown by pre-ranked gene-set enrichment analysis (GSEA).


b , Heatmap shows the up - regulated genes upon USP8 over - expression and the down - regulated genes upon USP8 depletion when compared with control MDA - MB - 231 cells by RNA - seq analysis ( left panel ) . qRT - PCR analysis of control or MDA - MB - 231 cells stably over - expressing USP8 ( USP8 OE ) or USP8 shRNA ( right panel ) .

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b, Heatmap shows the up-regulated genes upon USP8 over-expression and the down-regulated genes upon USP8 depletion when compared with control MDA-MB-231 cells by RNA-seq analysis (left panel). qRT-PCR analysis of control or MDA-MB-231 cells stably over-expressing USP8 (USP8 OE) or USP8 shRNA (right panel).


e , FACS analysis of EVs from 4T1 and MDA - MB - 231 cells infected with lentivirus encoding control shRNA ( Co . sh ) or USP8 shRNA .

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e, FACS analysis of EVs from 4T1 and MDA-MB-231 cells infected with lentivirus encoding control shRNA (Co.sh) or USP8 shRNA.


f , Immunoblot analysis of EVs from MDA - MB - 231 cells infected with lentivirus encoding control shRNA ( Co . sh ) or USP8 shRNA ( # 1 & # 2 ) .

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f, Immunoblot analysis of EVs from MDA-MB-231 cells infected with lentivirus encoding control shRNA (Co.sh) or USP8 shRNA (#1 & #2).


g , Immunoblot analysis of the EVs derived from MDA - MB - 231 cells ectopically expressing USP8 wt / cs .

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g, Immunoblot analysis of the EVs derived from MDA-MB-231 cells ectopically expressing USP8 wt/cs.


h , Experimental procedure in vivo : BALB / c mice were nipple injected with 4T1 - Luc cells ( 5 × 105 cells per mouse ) expressing control shRNA or doxycycline - inducible shRNA targeting USP8 ( shUSP8 ) and tumors were grown for 3 weeks , followed by the administration of doxycycline for 3 weeks ( n = 6 for each group ) . i , Quantification of the percentage of TβRII + crEVs in plasma samples from mice at day 24 . j , Immunoblot analysis of tumor and the TEVs ( left panel ) and quantification of the percentage of TβRII + EVs ( right panel ) in plasma samples from mice at day 42 . k , Normalized photon ﬂux at the indicated times . l ,

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

h, Experimental procedure in vivo: BALB/c mice were nipple injected with 4T1-Luc cells (5 × 105 cells per mouse) expressing control shRNA or doxycycline-inducible shRNA targeting USP8 (shUSP8) and tumors were grown for 3 weeks, followed by the administration of doxycycline for 3 weeks (n = 6 for each group). i, Quantification of the percentage of TβRII+ crEVs in plasma samples from mice at day 24. j, Immunoblot analysis of tumor and the TEVs (left panel) and quantification of the percentage of TβRII+ EVs (right panel) in plasma samples from mice at day 42. k, Normalized photon ﬂux at the indicated times. l,


l , Representative BLI signals of primary tumor in each group at day 42 after implantation of 4T1 cells ( 2 × 105 cells per mouse , n = 6 per group ) .

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l, Representative BLI signals of primary tumor in each group at day 42 after implantation of 4T1 cells (2 × 105 cells per mouse, n = 6 per group).


m , Normalized BLI signals ( left panel ) and bioluminescent images ( right panel ) of the lung metastasis nodules . Scale bars , 2 mm .

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m, Normalized BLI signals (left panel) and bioluminescent images (right panel) of the lung metastasis nodules. Scale bars, 2 mm.


n , Percentage of CD8 + T cells of tumor - draining lymph nodes ( TDLN ) at day 42 .

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n, Percentage of CD8+ T cells of tumor-draining lymph nodes (TDLN) at day 42.


b , Left : screening procedure for cellular components critical for the secretion of EV - TβRII ; Right : nanosight analysis of GFP positive EVs derived from MDA - MB - 231 cells expressing TβRII - GFP and pre - treated with anti - cancer compounds . n = 3 technical replicates .

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b, Left: screening procedure for cellular components critical for the secretion of EV-TβRII; Right: nanosight analysis of GFP positive EVs derived from MDA-MB-231 cells expressing TβRII-GFP and pre-treated with anti-cancer compounds. n = 3 technical replicates.


c , Immunoblot analysis of total cell lysates derived from MDA - MB - 231 breast cancer cell treated with USP8 inhibitor and TGF - β ( 2 . 5 ng / ml ) as indicated .

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c, Immunoblot analysis of total cell lysates derived from MDA-MB-231 breast cancer cell treated with USP8 inhibitor and TGF-β (2.5 ng/ml) as indicated.


d , Effect of USP8 inhibitor ( 5 μM ) on SMAD3 / SMAD4 - dependent CAGA12 - Luc transcriptional response induced by TGF - β ( 2 . 5 ng / ml ) for 16 h in HEK293T cells transfected with control vector ( Co . vec ) or USP8 wt / cs . n = 3 biological replicates .

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d, Effect of USP8 inhibitor (5 μM) on SMAD3/SMAD4-dependent CAGA12-Luc transcriptional response induced by TGF-β (2.5 ng/ml) for 16 h in HEK293T cells transfected with control vector (Co.vec) or USP8 wt/cs. n = 3 biological replicates.


e , Heatmap shows differentially expressed genes in control DMSO - treated or USP8 inhibitor ( 5 μM ) - treated MDA - MB - 231 cells for 24 h ( left panel ) . Volcano plot of transcriptome proﬁles between control and USP8 inhibitor treated MDA - MB - 231 cells ( right panel ) . Red and green dots represent genes significantly up - regulated and down - regulated in control MDA - MB - 231 cells .

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e, Heatmap shows differentially expressed genes in control DMSO-treated or USP8 inhibitor (5 μM)-treated MDA-MB-231 cells for 24 h (left panel). Volcano plot of transcriptome proﬁles between control and USP8 inhibitor treated MDA-MB-231 cells (right panel). Red and green dots represent genes significantly up-regulated and down-regulated in control MDA-MB-231 cells.


f , qRT - PCR analysis of control ( DMSO - treated ) or USP8 inhibitor ( 5 μM for 24 h ) treated MDA - MB - 231 cells . Relative mRNA levels are shown as a heatmap .

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f, qRT-PCR analysis of control (DMSO-treated) or USP8 inhibitor (5 μM for 24 h) treated MDA-MB-231 cells. Relative mRNA levels are shown as a heatmap.


g , Gene signatures indicating the up - regulated epithelial - mesenchymal - transition ( EMT ) ( upper panel ) and breast cancer progenitors - related gene set ( bottom panel ) are significantly enriched in control cells ; shown by pre - ranked gene - set enrichment analysis ( GSEA ) .

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g, Gene signatures indicating the up-regulated epithelial-mesenchymal-transition (EMT) (upper panel) and breast cancer progenitors-related gene set (bottom panel) are significantly enriched in control cells; shown by pre-ranked gene-set enrichment analysis (GSEA).


h , The down - regulated TGF - β signaling ( upper panel ) and metastasis - related gene sets ( bottom panel ) are significantly enriched in USP8 inhibitor - treated MDA - MB - 231 cells ; shown by pre - ranked gene - set enrichment analysis ( GSEA ) .

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h, The down-regulated TGF-β signaling (upper panel) and metastasis-related gene sets (bottom panel) are significantly enriched in USP8 inhibitor-treated MDA-MB-231 cells; shown by pre-ranked gene-set enrichment analysis (GSEA).


j , The total concentration of EVs by nanosight analysis ( left ) and the percentage of TβRII + EVs by FACS ( right ) from 4T1 and MDA - MB - 231 cells treated with USP8 inhibitor ( 5 μM ) for 24 h . n = 3 technical replicates .

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j, The total concentration of EVs by nanosight analysis (left) and the percentage of TβRII+ EVs by FACS (right) from 4T1 and MDA-MB-231 cells treated with USP8 inhibitor (5 μM) for 24 h. n = 3 technical replicates.


k - n , Experimental procedure in vivo MDA - MB - 231 - Luc cells ( 1 × 105 per mouse ) were intracardially injected into nude mice ( n = 7 for each group ) . USP8 inhibitor ( 1 mg / kg ) was injected at day 1 via intraperitoneal injection every the other day for three weeks . Normalized BLI signals ( left panel ) and BLI imaging of three representive nude mice from each group at week 6 ( right panel ) were shown ( k ) . Percentage of metastasis - free survival in each experimental group followed in time ( l ) . Number of bone metastasis nodules ( m ) . Percentage of TβRII + crEVs in plasma samples from mice ( left panel ) and immunoblot analysis of metastatic tumor nodule in mice and circulating EVs from plasma ( right panel ) ( n ) .

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k-n, Experimental procedure in vivo MDA-MB-231-Luc cells (1 × 105 per mouse) were intracardially injected into nude mice (n = 7 for each group). USP8 inhibitor (1 mg/kg) was injected at day 1 via intraperitoneal injection every the other day for three weeks. Normalized BLI signals (left panel) and BLI imaging of three representive nude mice from each group at week 6 (right panel) were shown (k). Percentage of metastasis-free survival in each experimental group followed in time (l). Number of bone metastasis nodules (m). Percentage of TβRII+ crEVs in plasma samples from mice (left panel) and immunoblot analysis of metastatic tumor nodule in mice and circulating EVs from plasma (right panel) (n).


( A ) Levels of KRAS , COUP - TFII , LDHA , and phosphorylated S6K ( T389 ) in KRASWT , KRASG12D , and KRAS - silenced KRASG12D MEF cells .

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(A) Levels of KRAS, COUP-TFII, LDHA, and phosphorylated S6K (T389) in KRASWT, KRASG12D, and KRAS-silenced KRASG12D MEF cells.


( B ) Extracellular acidification rate ( ECAR ) in KRASWT , KRAS G12D , and KRAS - or COUP - TFII - silenced KRAS G12D MEF cells .

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(B) Extracellular acidification rate (ECAR) in KRASWT, KRAS G12D, and KRAS- or COUP-TFII-silenced KRAS G12D MEF cells.


( C ) Lactate levels in MEF cells Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in KRASWT MEF . * * * p < 0 . 001 .

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(C) Lactate levels in MEF cells Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in KRASWT MEF. ***p<0.001.


( D ) Levels of COUP - TFII , LDHA , and phosphorylated S6K ( T389 ) in COUP - TFII - silenced KRASG12D MEF cells .

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(D) Levels of COUP-TFII, LDHA, and phosphorylated S6K (T389) in COUP-TFII-silenced KRASG12D MEF cells.


( A ) Levels of KRAS , COUP - TFII , LDHA , and phosphorylated S6K ( T389 ) in KRAS - silenced HCT116 and DLD - 1 cells .

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(A) Levels of KRAS, COUP-TFII, LDHA, and phosphorylated S6K (T389) in KRAS-silenced HCT116 and DLD-1 cells.


( B ) Levels of COUP - TFII , LDHA , and phosphorylated S6K ( T389 ) in COUP - TFII - silenced HCT116 and DLD - 1 cells .

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(B) Levels of COUP-TFII, LDHA, and phosphorylated S6K (T389) in COUP-TFII-silenced HCT116 and DLD-1 cells.


( C ) Effect of lactate on phosphorylated S6K ( T389 ) , 4E - BP1 ( T37 / 46 ) , ULK1 ( S757 ) in COUP - TFII - silenced HCT116 cells .

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(C) Effect of lactate on phosphorylated S6K (T389), 4E-BP1 (T37/46), ULK1 (S757) in COUP-TFII-silenced HCT116 cells.


( D ) Effects of COUP - TFII and the inhibition of LDHA on phosphorylated S6K ( T389 ) and Raptor ( S792 ) in KRAS - silenced HCT116 cells . Asterisk indicates exogenous HA - COUP - TFII . Arrow indicates phosphorylated Raptor ( S792 ) .

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(D) Effects of COUP-TFII and the inhibition of LDHA on phosphorylated S6K (T389) and Raptor (S792) in KRAS-silenced HCT116 cells. Asterisk indicates exogenous HA-COUP-TFII. Arrow indicates phosphorylated Raptor (S792).


( E ) Inhibition of COUP - TFII - silenced HCT116 , DLD - 1 , and MIA PaCa - 2 cell proliferation and its recovery by treatment with lactate . Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in control . * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 .

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(E) Inhibition of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cell proliferation and its recovery by treatment with lactate. Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in control. *p<0.05, **p<0.01, and ***p<0.001.


( F ) Clonogenic assay of COUP - TFII - silenced HCT116 , DLD - 1 , and MIA PaCa - 2 cells in the presence or absence of lactate .

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(F) Clonogenic assay of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cells in the presence or absence of lactate.


( A ) Effect of lactate on the ERK / TSC2 and AKT / TSC2 signaling pathway in serum - starved HCT116 cells .

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(A) Effect of lactate on the ERK/TSC2 and AKT/TSC2 signaling pathway in serum-starved HCT116 cells.


( B ) Effect of lactate on phosphorylated S6K ( T389 ) inhibited by TSC2 overexpression in HCT116 cells .

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(B) Effect of lactate on phosphorylated S6K (T389) inhibited by TSC2 overexpression in HCT116 cells.


( C ) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA - Rheb - overexpressing HCT116 cells after serum deprivation overnight .

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(C) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA-Rheb-overexpressing HCT116 cells after serum deprivation overnight.


( D ) Molecular interaction between TSC2 and recombinant GDP - loaded Rheb in the presence or absence of lactate in vitro .

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(D) Molecular interaction between TSC2 and recombinant GDP-loaded Rheb in the presence or absence of lactate in vitro.


( E ) Effect of lactate on Rheb activation in serum - starved HCT116 cells .

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(E) Effect of lactate on Rheb activation in serum-starved HCT116 cells.


( F ) Interaction between endogenous Rheb and mTOR in the presence or absence of lactate in HCT116 cells after serum deprivation overnight .

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(F) Interaction between endogenous Rheb and mTOR in the presence or absence of lactate in HCT116 cells after serum deprivation overnight.


( G ) Immunofluorescence analysis of staining for TSC2 and LAMP2 in the presence or absence of lactate in HCT116 cells after serum deprivation overnight .

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(G) Immunofluorescence analysis of staining for TSC2 and LAMP2 in the presence or absence of lactate in HCT116 cells after serum deprivation overnight.


( A ) Interaction between endogenous TSC2 and Rheb in the presence or absence of lactate in COUP - TFII - silenced HCT116 cells .

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(A) Interaction between endogenous TSC2 and Rheb in the presence or absence of lactate in COUP-TFII-silenced HCT116 cells.


( B ) Effect of lactate on Rheb activation in COUP - TFII - silenced HCT 116 cells .

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(B) Effect of lactate on Rheb activation in COUP-TFII-silenced HCT 116 cells.


( C ) Immunofluorescence analysis of staining for TSC2 and LAMP2 in HCT116 cells The degree of colocalization of TSC2 and LAMP2 is graphed as the mean ± SEM . * * * p < 0 . 001 .

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(C) Immunofluorescence analysis of staining for TSC2 and LAMP2 in HCT116 cells The degree of colocalization of TSC2 and LAMP2 is graphed as the mean ± SEM. ***p<0.001.


( D ) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA - Rheb - and / or COUP - TFII - overexpressing HCT116 cells treated with 25 nM trametinib for 24 h .

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(D) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA-Rheb- and/or COUP-TFII-overexpressing HCT116 cells treated with 25 nM trametinib for 24 h.


( E ) Effects of lactate and COUP - TFII overexpression on Rheb activation in HCT116 cells treated with 25 nM trametinib for 24 h .

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(E) Effects of lactate and COUP-TFII overexpression on Rheb activation in HCT116 cells treated with 25 nM trametinib for 24 h.


( F ) Immunofluorescence analysis of staining for TSC2 and LAMP2 in HCT116 cells The degree of colocalization of TSC2 and LAMP2 is graphed as the mean ± SEM . * * * p < 0 . 001 .

(F) Immunofluorescence analysis of staining for TSC2 and LAMP2 in HCT116 cells The degree of colocalization of TSC2 and LAMP2 is graphed as the mean ± SEM. ***p<0.001.


Effects of COUP - TFII inhibition and lactate treatment on tumor growth in vivo . ( A ) Growth curve in tumors derived from xenografted shScramble or shCOUP - TFII - HCT116 cells and treated with sodium chloride or sodium L - lactate ( 60 l x g - 1 body weight ; 150 mM ) .

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Effects of COUP-TFII inhibition and lactate treatment on tumor growth in vivo. (A) Growth curve in tumors derived from xenografted shScramble or shCOUP-TFII-HCT116 cells and treated with sodium chloride or sodium L-lactate (60 l x g-1 body weight; 150 mM).


( B ) tumor volume at the experimental endpoint , in tumors derived from xenografted shScramble or shCOUP - TFII - HCT116 cells and treated with sodium chloride or sodium L - lactate ( 60 l x g - 1 body weight ; 150 mM ) .

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(B) tumor volume at the experimental endpoint, in tumors derived from xenografted shScramble or shCOUP-TFII-HCT116 cells and treated with sodium chloride or sodium L-lactate (60 l x g-1 body weight; 150 mM).


Effects of COUP - TFII inhibition and lactate treatment on tumor growth in vivo . ( C ) Western blot analysis of the indicated proteins in tumors derived from xenografted shScramble or shCOUP - TFII - HCT116 cells and treated with sodium chloride or sodium L - lactate ( 60 l x g - 1 body weight ; 150 mM ) .

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Effects of COUP-TFII inhibition and lactate treatment on tumor growth in vivo. (C) Western blot analysis of the indicated proteins in tumors derived from xenografted shScramble or shCOUP-TFII-HCT116 cells and treated with sodium chloride or sodium L-lactate (60 l x g-1 body weight; 150 mM).


( D ) Effects of trametinib treatment and overexpression of COUP - TFII on tumor growth in vivo . Tumors were derived from xenografted HCT116 expressing HA - COUP - TFII or carrying a control vector and treated with trametinib ( 3mg / kg ) .

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(D) Effects of trametinib treatment and overexpression of COUP-TFII on tumor growth in vivo. Tumors were derived from xenografted HCT116 expressing HA-COUP-TFII or carrying a control vector and treated with trametinib (3mg/kg).


( E ) Western blot analysis of indicated proteins in tumors derived from xenografted HCT116 treated with trametinib with or without COUP - TFII overexpression . Asterisk indicates exogenous HA - COUP - TFII .

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(E) Western blot analysis of indicated proteins in tumors derived from xenografted HCT116 treated with trametinib with or without COUP-TFII overexpression. Asterisk indicates exogenous HA-COUP-TFII.


E Irf1 expression in human blastocysts . Integrated single cell RNA - seq data from blastocysts collected at or cultured to indicated time points . Data is derived from a total of 16 embryos which passed quality control metrics .

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E Irf1 expression in human blastocysts. Integrated single cell RNA-seq data from blastocysts collected at or cultured to indicated time points. Data is derived from a total of 16 embryos which passed quality control metrics.


A Western Blot analysis of Irf1 - / - in ESC and EpiLC , probed with antibodies against IRF1 and VINCULIN as loading control . Shown are two independent clonal cell lines .

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A Western Blot analysis of Irf1-/- in ESC and EpiLC, probed with antibodies against IRF1 and VINCULIN as loading control. Shown are two independent clonal cell lines.


C . Karyograms of trisomic ES cells shows that these cell lines have extra copies of chromosomes 6 , 8 , 11 , or 15 . The Ts6 and Ts11 ES cells harbored isochromosomes of the two identical trisomic chromosomes . Ts , trisomy .

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C. Karyograms of trisomic ES cells shows that these cell lines have extra copies of chromosomes 6, 8, 11, or 15. The Ts6 and Ts11 ES cells harbored isochromosomes of the two identical trisomic chromosomes. Ts, trisomy.


D . CGH analysis of trisomic ES cell lines . The relative gene copy number is plotted using a log2 scale .

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D. CGH analysis of trisomic ES cell lines. The relative gene copy number is plotted using a log2 scale.


E . CGH analysis of the genome stability of trisomic ES cells .

E. CGH analysis of the genome stability of trisomic ES cells.


F . CGH analysis of the genome integrity of trisomic ES cell subclones .

F. CGH analysis of the genome integrity of trisomic ES cell subclones.


A . Cumulative growth curves of trisomic and wild - type ES cells . The number of ES cells was counted at the indicated days in culture . The cell lines were tested in triplicates . Error bars , ± S . D . * P < 0 . 05 , * * P < 0 . 01 .

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A. Cumulative growth curves of trisomic and wild-type ES cells. The number of ES cells was counted at the indicated days in culture. The cell lines were tested in triplicates. Error bars, ±S.D. * P < 0.05, ** P < 0.01.


B . Cell cycle distribution of trisomic and wild - type ES cell lines . The percentage of cells in G1 , S , and G2 / M phases was determined via flow cytometry . Error bars , ± S . D . n = 3 . * P < 0 . 05 , * * P < 0 . 01 .

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B. Cell cycle distribution of trisomic and wild-type ES cell lines. The percentage of cells in G1, S, and G2/M phases was determined via flow cytometry. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.


C . The percentage of Annexin V + apoptotic cells was quantified . Error bars , ± S . D . n = 3 .

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C. The percentage of Annexin V+ apoptotic cells was quantified. Error bars, ±S.D. n=3.


D . Trisomic and wild - type ES cells were treated with Cycloheximide or MG132 for 48 hours at the indicated doses . Error bars , ± S . D . n = 3 . * P < 0 . 05 , * * P < 0 . 01 .

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D. Trisomic and wild-type ES cells were treated with Cycloheximide or MG132 for 48 hours at the indicated doses. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.


A . The coloning - formation rates of the trisomic and wild - type ES cells . Error bars , ± S . D . n = 3 . * P < 0 . 05 , * * P < 0 . 01 .

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A. The coloning-formation rates of the trisomic and wild-type ES cells. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.


B . Alkaline phosphatase ( AP ) staining of trisomic and wild - type ES cells after LIF withdrawal ( LIF 0 U / ml ) for 5 days . Scale bar , 500 μm . C . Evaluation of AP positive and negative colonies after LIF withdrawal . Black bars , undifferentiated colonies ; orange bars , partially differentiated colonies ; green bars , totally differentiated colonies . Error bars , ± S . D . n = 3 . * * , P < 0 . 01 .

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B. Alkaline phosphatase (AP) staining of trisomic and wild-type ES cells after LIF withdrawal (LIF 0 U/ml) for 5 days. Scale bar, 500 μm.C. Evaluation of AP positive and negative colonies after LIF withdrawal. Black bars, undifferentiated colonies; orange bars, partially differentiated colonies; green bars, totally differentiated colonies. Error bars, ±S.D. n=3. **, P < 0.01.


D . Boxplots of EB diameter at the indicated days of EB formation . Each boxplot was plotted for more than 20 EBs . * P < 0 . 05 , * * P < 0 . 01 .

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D. Boxplots of EB diameter at the indicated days of EB formation. Each boxplot was plotted for more than 20 EBs. * P < 0.05, ** P < 0.01.


E . Time course analysis of marker genes expression during EB formation by RT - qPCR . Error bars , ± S . D . n = 3 .

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E. Time course analysis of marker genes expression during EB formation by RT-qPCR. Error bars, ±S.D. n=3.


F . Quantification of Oct4 , Nanog and Rex1 expression in trisomic and wild - type ES cell derived EBs at day 8 of differentiation . Error bars , ± S . D . n = 3 . * * P < 0 . 01 .

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F. Quantification of Oct4, Nanog and Rex1 expression in trisomic and wild-type ES cell derived EBs at day 8 of differentiation. Error bars, ±S.D. n=3. ** P < 0.01.


A . Teratomagrowth curves of trisomic and wild - type ES cells after subcutaneous injection into SCIDmice . 1 × 106 cells were injected per site ( 6 sites / per group ) . Error bars , ± S . D . * P < 0 . 05 .

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A. Teratomagrowth curves of trisomic and wild-type ES cells after subcutaneous injection into SCIDmice. 1×106 cells were injected per site (6 sites/per group). Error bars, ±S.D. * P < 0.05.


B . Four - week teratomas derived from wild - type and trisomic ( Ts8 or Ts11 ) ES cell lines . 5 × 105 cells were injected per site ( 8 sites / per group ) .

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B. Four-week teratomas derived from wild-type and trisomic (Ts8 or Ts11) ES cell lines. 5×105 cells were injected per site (8 sites/per group).


C . Representative images of undifferentiated regions in Ts8 ES cell derived teratomas ( middle , top and bottom ) and the primitive neural rosettes in the teratomas derived from Ts15 ES cells ( right , top and bottom ) . The images of mature neuroectoderm from wild - type ES cell formed teratomas ( left , top and bottom ) were shown . The bottom panels were the enlarged views of the white dotted boxes of the top panels . Hematoxylin and eosin staining . Scale bars , 50 μm .

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C. Representative images of undifferentiated regions in Ts8 ES cell derived teratomas (middle, top and bottom) and the primitive neural rosettes in the teratomas derived from Ts15 ES cells (right, top and bottom). The images of mature neuroectoderm from wild-type ES cell formed teratomas (left, top and bottom) were shown. The bottom panels were the enlarged views of the white dotted boxes of the top panels. Hematoxylin and eosin staining. Scale bars, 50 μm.


D . Immunohistochemistry analysis of Oct4 expression in wild - type , Homo - 11 , Ts6 , Ts8 , Ts11 , or Ts15 ES cellteratomas produced via the injection of 5 × 105 ES cells . Scale bar , 100 μm .

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D. Immunohistochemistry analysis of Oct4 expression in wild-type, Homo-11, Ts6, Ts8, Ts11, or Ts15 ES cellteratomas produced via the injection of 5×105 ES cells. Scale bar, 100 μm.


E . AP staining of the primary cultured teratomacells derived from trisomic and wild - type ES cells . Ts6 , Ts8 , or Ts11 ESC teratomacells grew ES - like colonies that were stained positive for AP . Scale bar , 200 μm .

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E. AP staining of the primary cultured teratomacells derived from trisomic and wild-type ES cells. Ts6, Ts8, or Ts11 ESC teratomacells grew ES-like colonies that were stained positive for AP. Scale bar, 200 μm.


F . The expression of marker genes in cultured teratoma cells ( p3 ) . Error bars , ± S . D . n = 3 . * * P < 0 . 01 .

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F. The expression of marker genes in cultured teratoma cells (p3). Error bars, ±S.D. n=3. ** P < 0.01.


B . Karyograms of Di8 , Di11 and Di15 ES cell lines . Di8 was the corrected ES cell line from Ts8 , showing diploid karyotype and Y chromosome loss . Di11 and Di15 were from Ts11 and Ts15 ES cells respectively .

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B. Karyograms of Di8, Di11 and Di15 ES cell lines. Di8 was the corrected ES cell line from Ts8, showing diploid karyotype and Y chromosome loss. Di11 and Di15 were from Ts11 and Ts15 ES cells respectively.


C . AP staining of trisomic and the corrected diploid ES cells after LIF withdrawal ( LIF 0 U / ml ) for 5 days . Scale bar , 500 μm .

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C. AP staining of trisomic and the corrected diploid ES cells after LIF withdrawal (LIF 0 U/ml) for 5 days. Scale bar, 500 μm.


D . Teratomagrowth curves of trisomic and the corrected diploid ES cells . The injected cell number was 5 × 105 / site ( 8 sites / per group ) . Error bars , ± S . D . * * P < 0 . 01 .

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D. Teratomagrowth curves of trisomic and the corrected diploid ES cells. The injected cell number was 5×105/site (8 sites/per group). Error bars, ±S.D. ** P < 0.01.


E . Scatterplot of four - week teratoma weight . The teratomas were formed by the corrected diploid ES cells and their paternal trisomic ES cells . * P < 0 . 05 .

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E. Scatterplot of four-week teratoma weight. The teratomas were formed by the corrected diploid ES cells and their paternal trisomic ES cells. * P < 0.05.


F . RT - qPCR analysis of some gene expression in trisomic and wild - type ES cells . Error bars , ± S . D . n = 3 . * P < 0 . 05 , * * P < 0 . 01 .

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F. RT-qPCR analysis of some gene expression in trisomic and wild-type ES cells. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.


A . Morphology of EBs grew for 8 days derived from individual or mixed ES cell lines . Hematoxylin and eosin staining . Scale bar , 100 μm .

A. Morphology of EBs grew for 8 days derived from individual or mixed ES cell lines. Hematoxylin and eosin staining. Scale bar, 100 μm.


B . Quantification of Nanog expression in trisomic , wild - type , and mixed ES cell - derived EBs cultured for 8 days . Error bars , ± S . D . n = 3 . * * P < 0 . 01 .

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B. Quantification of Nanog expression in trisomic, wild-type, and mixed ES cell-derived EBs cultured for 8 days. Error bars, ±S.D. n=3. ** P < 0.01.


C . The morphology of EBs at day 10 of differentiation . M15 was the normal EB formation condition and was used as control . Black arrows indicate the cystic structures .

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C. The morphology of EBs at day 10 of differentiation. M15 was the normal EB formation condition and was used as control. Black arrows indicate the cystic structures.


D . Boxplots of EBs diameter at day 10 of EB formation under M15 , CM , and M15 supplemented with Bmp4 conditions . Each boxplot was plotted for more than 10 EBs . EBs cultured under CM or Bmp4 condition were compared to EBs of the same cell line cultured under M15 condition . * P < 0 . 05 , * * P < 0 . 01 .

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D. Boxplots of EBs diameter at day 10 of EB formation under M15, CM, and M15 supplemented with Bmp4 conditions. Each boxplot was plotted for more than 10 EBs. EBs cultured under CM or Bmp4 condition were compared to EBs of the same cell line cultured under M15 condition. * P < 0.05, ** P < 0.01.


RNAi was induced with an empty vector as a negative control ( panel I ) , whereas a smg - 2 clone ( panel II ) was used as a positive control . Panels i and ii show brightfield images of the PTCxi strain treated with the negative and positive controls , respectively . Depletion of five genes ( panels III to VII ) resulted in increased GFP expression . Panels iii to vii show brightfield images of the phenotypes of the affected worms . The scale bars correspond to 100 μm .

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RNAi was induced with an empty vector as a negative control (panel I), whereas a smg-2 clone (panel II) was used as a positive control. Panels i and ii show brightfield images of the PTCxi strain treated with the negative and positive controls, respectively. Depletion of five genes (panels III to VII) resulted in increased GFP expression. Panels iii to vii show brightfield images of the phenotypes of the affected worms. The scale bars correspond to 100 μm.


Depletion of the novel NMD genes in C . elegans leads to upregulation of the PTCx NMD reporter mRNA , which was monitored by quantitative RT - PCR relative to the expression of ama - 1 reference gene . The values shown are the average fold - change ( mean ± SEM ) from at least three independent experiments relative to empty vector - depleted worms . Statistical analysis was performed using the Mann - Whitney U - test for non - parametric distributions . * P < 0 . 05 .

Depletion of the novel NMD genes in C. elegans leads to upregulation of the PTCx NMD reporter mRNA, which was monitored by quantitative RT-PCR relative to the expression of ama-1 reference gene. The values shown are the average fold-change (mean ± SEM) from at least three independent experiments relative to empty vector-depleted worms. Statistical analysis was performed using the Mann-Whitney U-test for non-parametric distributions. *P < 0.05.


A - C Embryos expressing a GFP - NMD sensor ( green ) under the control of nompA regulatory sequences show signal in support cells linked to the embryonic peripheral nervous system ( PNS ) [ labeled by 22C10 signal ( magenta ) ] . DAPI signal is shown in blue . Expression of UAS - RNAi constructs against Upf1 ( B ) or nompA ( C ) genes using a nompA - Gal4 driver leads to upregulation of the GFP - NMD sensor when compared to wild - type ( no RNAi ) ( A ) , revealing a reduction in NMD activity in the knockdown conditions . Scale bars represent 100 μm . D - F Higher magnification ( 40 × ) of the areas marked by a rectangle in ( A - C ) further illustrates the upregulation of the GFP - NMD sensor in Upf1 - ( E ) and NompA - depleted cells ( F ) . Optical fields include embryonic abdominal segments A4 - A5 . Scale bars represent 10 μm .

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A-C Embryos expressing a GFP-NMD sensor (green) under the control of nompA regulatory sequences show signal in support cells linked to the embryonic peripheral nervous system (PNS) [labeled by 22C10 signal (magenta)]. DAPI signal is shown in blue. Expression of UAS-RNAi constructs against Upf1 (B) or nompA (C) genes using a nompA-Gal4 driver leads to upregulation of the GFP-NMD sensor when compared to wild-type (no RNAi) (A), revealing a reduction in NMD activity in the knockdown conditions. Scale bars represent 100 μm.D-F Higher magnification (40×) of the areas marked by a rectangle in (A-C) further illustrates the upregulation of the GFP-NMD sensor in Upf1- (E) and NompA-depleted cells (F). Optical fields include embryonic abdominal segments A4-A5. Scale bars represent 10 μm.


G Quantification of GFP signal in cells marked by an arrow in panels ( D - F ) shows a significant upregulation of NMD sensor expression upon downregulation of Upf1 ( dark gray ) and NompA ( black ) compared to wild - type ( no RNAi ) ( light gray ) . Results represent the average of five biological replicates ( mean ± SEM ) . Pair - wise comparisons were performed using a one - tailed t - test ( non - parametric ) between treatments and wild - type . * * * P < 0 . 001 .

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G Quantification of GFP signal in cells marked by an arrow in panels (D-F) shows a significant upregulation of NMD sensor expression upon downregulation of Upf1 (dark gray) and NompA (black) compared to wild-type (no RNAi) (light gray). Results represent the average of five biological replicates (mean ± SEM). Pair-wise comparisons were performed using a one-tailed t-test (non-parametric) between treatments and wild-type. ***P < 0.001.


A , B HeLa cells stably expressing a wild - type β - globin reporter ( A ) or a β - globin NS39 NMD reporter ( B ) were mock - depleted or depleted of UPF2 , GNL2 , SEC13 , RAB27A , RAB27B or both RAB27A and RAB27B . The level of the β - globin mRNA was monitored by quantitative RT - PCR relative to two reference genes ( POLR2J and ACTB ) . The values shown are the average fold - change ( mean ± SEM ) from four independent experiments relative to mock - depleted cells ( control ) . Statistical analysis was performed using the Mann - Whitney U - test for non - parametric distributions . * P < 0 . 05 . The level of depletion of NMD factors is shown in Supplementary Fig S3C .

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A, B HeLa cells stably expressing a wild-type β-globin reporter (A) or a β-globin NS39 NMD reporter (B) were mock-depleted or depleted of UPF2, GNL2, SEC13, RAB27A, RAB27B or both RAB27A and RAB27B. The level of the β-globin mRNA was monitored by quantitative RT-PCR relative to two reference genes (POLR2J and ACTB). The values shown are the average fold-change (mean ± SEM) from four independent experiments relative to mock-depleted cells (control). Statistical analysis was performed using the Mann-Whitney U-test for non-parametric distributions. *P < 0.05. The level of depletion of NMD factors is shown in Supplementary Fig S3C.


C , D Analysis of the half - life of β - globin reporters . Samples were collected at the indicated time points , and the mRNA levels of the HBB reporters were monitored by qRT - PCR and normalized to POLR2J and ACTB reference genes . The values shown are the average fold - change ( mean ± SEM ) from three independent experiments relative to the first time point .

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C, D Analysis of the half-life of β-globin reporters. Samples were collected at the indicated time points, and the mRNA levels of the HBB reporters were monitored by qRT-PCR and normalized to POLR2J and ACTB reference genes. The values shown are the average fold-change (mean ± SEM) from three independent experiments relative to the first time point.


E Depletion of GNL2 and SEC13 leads to a significant upregulation in the mRNA levels of endogenous NMD substrates . Samples were analyzed as described in ( A , B ) * P < 0 . 05 ; * * * P < 0 . 01 , * * * P < 0 . 001 .

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E Depletion of GNL2 and SEC13 leads to a significant upregulation in the mRNA levels of endogenous NMD substrates. Samples were analyzed as described in (A, B) *P < 0.05; ***P < 0.01, ***P < 0.001.


F GNL2 and SEC13 contribute to the negative NMD feedback loop , regulating the levels of transcripts encoding NMD factors . RT - qPCR analysis of total cellular RNA from HeLa cells depleted of GNL2 ( in green ) and SEC13 ( in purple ) is shown . The graph shows distribution of relative fold - change from eight independent experiments relative to mock - depleted cells ( control ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 .

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F GNL2 and SEC13 contribute to the negative NMD feedback loop, regulating the levels of transcripts encoding NMD factors. RT-qPCR analysis of total cellular RNA from HeLa cells depleted of GNL2 (in green) and SEC13 (in purple) is shown. The graph shows distribution of relative fold-change from eight independent experiments relative to mock-depleted cells (control). Statistical analysis was performed using Student's t-test. *P < 0.05.


A Peptide dotblot array probed with anti - H3K122succ AB # 2 . Peptides with indicated modifications were spotted on membranes .

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A Peptide dotblot array probed with anti-H3K122succ AB #2. Peptides with indicated modifications were spotted on membranes.


B Immunoblot analysis with anti - H3K122succ AB # 2 on indicated histone octamers . Octamers with H3K122succ are specifically recognized .

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B Immunoblot analysis with anti-H3K122succ AB #2 on indicated histone octamers. Octamers with H3K122succ are specifically recognized.


C Pairwise scatterplots of H3K122succ read counts within peaks from ChIP experiments using all three H3K122succ antibodies ( AB # 1 , AB # 2 and AB # 3 ) .

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C Pairwise scatterplots of H3K122succ read counts within peaks from ChIP experiments using all three H3K122succ antibodies (AB #1, AB #2 and AB #3).


A IGV browser snapshots showing the distribution of reads around the TSS of the actively transcribed PPM1D gene ( chr17 : 60 , 587 , 313 - 60 , 643 , 064 ; top panel ) and of the inactive CD40 gene ( chr20 : 46 , 114 , 180 - 46 , 129 , 721 ; bottom panel ) for the indicated histone modifications , input and RNAseq .

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A IGV browser snapshots showing the distribution of reads around the TSS of the actively transcribed PPM1D gene (chr17:60,587,313-60,643,064; top panel) and of the inactive CD40 gene (chr20:46,114,180-46,129,721; bottom panel) for the indicated histone modifications, input and RNAseq.


F H3K122succ occupancy profile over a meta gene as a function of expression level ( based on RNA - seq quartiles : Q1 - least expressed , Q4 - most expressed ) . All genes were normalized to the same length .

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F H3K122succ occupancy profile over a meta gene as a function of expression level (based on RNA-seq quartiles: Q1 - least expressed, Q4 - most expressed). All genes were normalized to the same length.


G Boxplots visualizing expression values of genes ( reads per kb ; RPK ) with peaks for indicated different combinations of histone PTMs at their TSS . Histone modifications are ( from left to right ) : none ( no PTM , black ) ; only H3K122succ ( green ) ; H3K122succ plus one tail modification ( H3K4me3 , or H3K9ac , or H3K27ac , green and light orange ) ; H3K122succ plus two tail modifications ( green , yellow and light orange ) ; H3K4me3 and H3K9ac and H3K27ac without H3K122succ ( yellow , light orange and dark orange ) ; H3K122succ and all three tail modifications ( green , yellow , light orange , and dark orange ) . The number of genes within the groups is indicated below each boxplot . Boxes indicate the range between the first and third quartile , the central line depicts the median , and the whiskers span the range of the data while extending no further than 1 . 5 times the interquartile range . Each distribution was compared to the " no PTM " distribution .

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G Boxplots visualizing expression values of genes (reads per kb; RPK) with peaks for indicated different combinations of histone PTMs at their TSS. Histone modifications are (from left to right): none (no PTM, black); only H3K122succ (green); H3K122succ plus one tail modification (H3K4me3, or H3K9ac, or H3K27ac, green and light orange); H3K122succ plus two tail modifications (green, yellow and light orange); H3K4me3 and H3K9ac and H3K27ac without H3K122succ (yellow, light orange and dark orange); H3K122succ and all three tail modifications (green, yellow, light orange, and dark orange). The number of genes within the groups is indicated below each boxplot. Boxes indicate the range between the first and third quartile, the central line depicts the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Each distribution was compared to the "no PTM" distribution.


A H3K122succ levels ( detected by immunoblot ) , relative to the scramble siRNA control , upon depletion of indicated HAT enzyme ( s ) ( GCN5 , pCAF , p300 and / or CBP ) from MCF7 cells by siRNA . The bar graph shows a representative

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A H3K122succ levels (detected by immunoblot), relative to the scramble siRNA control, upon depletion of indicated HAT enzyme(s) (GCN5, pCAF, p300 and/or CBP) from MCF7 cells by siRNA. The bar graph shows a representative experiment


B In vitro succinyltransferase assay on recombinant histone octamers as substrate . After incubation with p300 and suc - CoA , the H3K122succ levels were assessed by immunoblot . Ponceau staining of membrane is shown as a loading control .

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B In vitro succinyltransferase assay on recombinant histone octamers as substrate. After incubation with p300 and suc-CoA, the H3K122succ levels were assessed by immunoblot. Ponceau staining of membrane is shown as a loading control.


C In vitro succinyltransferase assay on unmodified peptides spanning H3K122 . Samples were incubated with increasing amounts of p300 ( 0 to 160 ng ) in the presence of radiolabeled suc - CoA . The radioactivity incorporated was quantified by liquid scintillation counting . The plot displays average counts per minute ( CPMs ) , ± SD and individual datapoints of two independent replicates .

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C In vitro succinyltransferase assay on unmodified peptides spanning H3K122. Samples were incubated with increasing amounts of p300 (0 to 160 ng) in the presence of radiolabeled suc-CoA. The radioactivity incorporated was quantified by liquid scintillation counting. The plot displays average counts per minute (CPMs), ± SD and individual datapoints of two independent replicates.


In vitro desuccinylation assay on H3K122succ peptides with recombinant SIRT7 ( D ) Unmodified H3K122 peptide was used as control ( left dot ) . Peptides were probed with H3K122succ AB # 2 after incubation with sirtuins .

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In vitro desuccinylation assay on H3K122succ peptides with recombinant SIRT7 (D) Unmodified H3K122 peptide was used as control (left dot). Peptides were probed with H3K122succ AB #2 after incubation with sirtuins.


In vitro desuccinylation assay on H3K122succ peptides with recombinant SIRT5 ( E ) . Unmodified H3K122 peptide was used as control ( left dot ) . Peptides were probed with H3K122succ AB # 2 after incubation with sirtuins .

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In vitro desuccinylation assay on H3K122succ peptides with recombinant SIRT5 (E). Unmodified H3K122 peptide was used as control (left dot). Peptides were probed with H3K122succ AB #2 after incubation with sirtuins.


F H3K122succ levels in Sirt5 - KO cells . Histones were acid extracted from wild type ( WT ) and Sirt5 - KO MEFs and the levels of H3K122succ determined by immunoblot . The level of H3K122succ was normalized to histones extracted from WT MEFs .

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F H3K122succ levels in Sirt5-KO cells. Histones were acid extracted from wild type (WT) and Sirt5-KO MEFs and the levels of H3K122succ determined by immunoblot. The level of H3K122succ was normalized to histones extracted from WT MEFs.


B In vitro transcription in the presence of GAL4 - VP16 and p300 , in the presence of no CoA , ac - CoA or suc - CoA . Shown is a representative autoradiogram of the IVT product . C Quantification of IVT reactions . Expression relative to no CoA is plotted .

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B In vitro transcription in the presence of GAL4-VP16 and p300, in the presence of no CoA, ac-CoA or suc-CoA. Shown is a representative autoradiogram of the IVT product. C Quantification of IVT reactions. Expression relative to no CoA is plotted.


D In vitro transcription on unmodified chromatin or chromatin site specifically succinylated at H3K122 . Shown is a representative autoradiogram . E Quantification of IVT reactions . Expression relative to transcription on unmodified chromatin is plotted .

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D In vitro transcription on unmodified chromatin or chromatin site specifically succinylated at H3K122. Shown is a representative autoradiogram. E Quantification of IVT reactions. Expression relative to transcription on unmodified chromatin is plotted.


A Schematic representation of the smFRET assay ( top ) , as well as merged smFRET efficiency histograms of unmodified ( middle ) and H3K122succ mono - nucleosomes ( bottom ) measured under indicated NaCl conditions ( color coded ) . The nucleosome population is highlighted by the dashed box .

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A Schematic representation of the smFRET assay (top), as well as merged smFRET efficiency histograms of unmodified (middle) and H3K122succ mono-nucleosomes (bottom) measured under indicated NaCl conditions (color coded). The nucleosome population is highlighted by the dashed box.


B Fraction of bursts in the nucleosome population ( FRET efficiency , E ≥ 0 . 45 ) for unmodified ( red , open circles ) and H3K122succ nucleosomes ( turquoise , open circles ) plotted against NaCl concentrations and normalized to the respective mean at 0 M NaCl . Mean values ( filled circles ) were fitted with the Boltzmann sigmoid function ( solid lines ) to determine the NaCl concentration where half of the nucleosomes have disassembled ( x0 ) as 0 . 61 M ( ± 0 . 04 ) for unmodified and 0 . 49 M ( ± 0 . 03 ) for H3K122succ nucleosomes .

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B Fraction of bursts in the nucleosome population (FRET efficiency, E ≥ 0.45) for unmodified (red, open circles) and H3K122succ nucleosomes (turquoise, open circles) plotted against NaCl concentrations and normalized to the respective mean at 0 M NaCl. Mean values (filled circles) were fitted with the Boltzmann sigmoid function (solid lines) to determine the NaCl concentration where half of the nucleosomes have disassembled (x0) as 0.61 M (± 0.04) for unmodified and 0.49 M (± 0.03) for H3K122succ nucleosomes.


AGS cells were infected with H . pylori or treated with 20 ng / ml LTα1β2 for the indicated times . Total cell lysates were analyzed by immunoblotting using the indicated antibodies .

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AGS cells were infected with H. pylori or treated with 20 ng/ml LTα1β2 for the indicated times. Total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS cells were infected with H . pylori P1 wild ‑ type or isogenic ∆ HP0857 ( mutated in the gmhA gene ) strains . Total cell lysates were analyzed by immunoblotting using the indicated antibodies .

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AGS cells were infected with H. pylori P1 wild‑type or isogenic ∆HP0857 (mutated in the gmhA gene) strains. Total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS cells were treated with 200 nM synthetic ADP ‑ heptose for the indicated times . Total cell lysates were analyzed by immunoblotting using the indicated antibodies .

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AGS cells were treated with 200 nM synthetic ADP‑heptose for the indicated times. Total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS cells were pre ‑ incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by H . pylori infection or addition of ADP - heptose ( 200 nM ) . Total cell lysates were analyzed by immunoblotting using the indicated antibodies . CagA demonstrated a similar infection rate .

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AGS cells were pre‑incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by H. pylori infection or addition of ADP-heptose (200 nM). Total cell lysates were analyzed by immunoblotting using the indicated antibodies. CagA demonstrated a similar infection rate.


Parental AGS , ALPK1 ‑ KO and TIFA ‑ KO cells were infected with H . pylori for the times shown . Total cell lysates were analyzed by immunoblotting using the indicated antibodies . CagA demonstrated a similar infection rate of the different cells . Asterisk denotes an unspecific band and the arrow indicates the band corresponding to phosphorylated p100 .

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Parental AGS, ALPK1‑KO and TIFA‑KO cells were infected with H. pylori for the times shown. Total cell lysates were analyzed by immunoblotting using the indicated antibodies. CagA demonstrated a similar infection rate of the different cells. Asterisk denotes an unspecific band and the arrow indicates the band corresponding to phosphorylated p100.


Total RNA was isolated from ALPK1 ‑ KO cells and analyzed using quantitative RT ‑ PCR for the ALPK1 transcript . Data shown depict the average of triplicate determinations normalized to the RPL13A housekeeping gene . Error bars denote RQ ± RQmin / RQmax .

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Total RNA was isolated from ALPK1‑KO cells and analyzed using quantitative RT‑PCR for the ALPK1 transcript. Data shown depict the average of triplicate determinations normalized to the RPL13A housekeeping gene. Error bars denote RQ ± RQmin/RQmax.