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 .

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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 .

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

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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 ) .

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 ) .

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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). 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 ) .

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 ,

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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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 ) .

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

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

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 ) .

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

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).


B , C , Representative images of pPLT3 : : erCFP ( cyan ) expressing and PI - stained ( red ) Arabidopsis roots in Col or wox5 background , respectively . D , Mean fluorescence intensities of the pPLT3 : : erCFP roots summarized in box and scatter plots . The mean fluorescence intensity of the CFP signal in Col roots was to set to 100 % . D , Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , □ = mean value , = minimum / maximum . The data was statistically analyzed by one - way ANOVA and Holm - Sidak post - hoc multiple comparisons test . Asterisks indicate statistically significant differences ( α = 0 . 01 ) . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype and results from two technical replicates . B , C , Scale bars represent 10 µm . PI = propidium iodide CFP = cyan fluorescent protein .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative images of pPLT3::erCFP (cyan) expressing and PI-stained (red) Arabidopsis roots in Col or wox5 background, respectively. D, Mean fluorescence intensities of the pPLT3::erCFP roots summarized in box and scatter plots. The mean fluorescence intensity of the CFP signal in Col roots was to set to 100 %. D, Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. The data was statistically analyzed by one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test. Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from two technical replicates. B, C, Scale bars represent 10 µm. PI = propidium iodide CFP = cyan fluorescent protein.


E , F , Representative images of pPLT3 : : PLT3 - YFP ( yellow ) expressing and FM4 - 64 - stained ( red ) Arabidopsis roots in Col or wox5 mutant background , respectively . g , Mean fluorescence intensities of the pPLT3 : : PLT3 - YFP expressing roots summarized in box and scatter plots . The mean fluorescence intensity of the YFP signal in Col roots was to set to 100 % . G , Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , □ = mean value , = minimum / maximum . The data was statistically analyzed by one - way ANOVA and Holm - Sidak post - hoc multiple comparisons test . Asterisks indicate statistically significant differences ( α = 0 . 01 ) . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype and results from two technical replicates . , E , F , Scale bars represent 10 µm . YFP = yellow fluorescent

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative images of pPLT3::PLT3-YFP (yellow) expressing and FM4-64-stained (red) Arabidopsis roots in Col or wox5 mutant background, respectively. g, Mean fluorescence intensities of the pPLT3::PLT3-YFP expressing roots summarized in box and scatter plots. The mean fluorescence intensity of the YFP signal in Col roots was to set to 100%. G, Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. The data was statistically analyzed by one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test. Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from two technical replicates. , E, F, Scale bars represent 10 µm. YFP = yellow fluorescent protein


A - F , Representative FM4 - 64 - stained Arabidopsis roots ( grey ) expressing pWOX5 : : mVenus - NLS ( green ) in Col , plt2 , plt3 and plt2 , plt3 double mutant background in longitudinal ( A - D ) , or transversal ( E - F ) optical sections . E ' , F ' , Analysis of representative images in ( E ) and ( F ) in Imaris to detect and count individual expressing nuclei . E ' ' , F ' ' , Overlay of 10 roots ( biological replicates ) showing the area of detected fluorescence ( high levels in red , low levels in blue ) in Col and plt2 , plt3 double mutant roots . G , Number of nuclei ( biological replicates ) expressing pWOX5 : : mVenus - NLS in Col and plt2 , plt3 double mutant roots summarized in box and scatter plots . H , Area of WOX5 expression in µm2 in Col and plt2 , plt3 double mutant roots summarized in box and scatter plots . G , H Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , □ = mean value , = minimum / maximum . ( G , H ) Kruskal - Wallis ANOVA analysis with subsequent Dunns test ( G ) or one - way ANOVA and post - hoc Holm - Sidak multiple comparisons test was used to test for statistical significance ( H ) . Asterisks indicate statistically significant differences ( α = 0 . 01 ) . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype and results from three technical replicates per genotype . Scale bars represent 10 µm ; NLS = nuclear localisation signal .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F, Representative FM4-64-stained Arabidopsis roots (grey) expressing pWOX5::mVenus-NLS (green) in Col, plt2, plt3 and plt2, plt3 double mutant background in longitudinal (A-D), or transversal (E-F) optical sections. E', F', Analysis of representative images in (E) and (F) in Imaris to detect and count individual expressing nuclei. E'', F'', Overlay of 10 roots (biological replicates) showing the area of detected fluorescence (high levels in red, low levels in blue) in Col and plt2, plt3 double mutant roots. G, Number of nuclei (biological replicates) expressing pWOX5::mVenus-NLS in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. H, Area of WOX5 expression in µm2 in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. G, H Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. (G, H) Kruskal-Wallis ANOVA analysis with subsequent Dunns test (G) or one-way ANOVA and post-hoc Holm-Sidak multiple comparisons test was used to test for statistical significance (H). Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from three technical replicates per genotype. Scale bars represent 10 µm; NLS = nuclear localisation signal.


A , Schematic representation of a longitudinal section of an Arabidopsis RM . QC cells are marked in red , CSCs are marked in dark blue , CCs in light blue . Combined mPSPI ( grey ) and EdU ( red ) staining for 24 hours ( SCN staining ) to analyse the CSC ( A - I ) and QC division phenotype ( J - R ) within the same roots are shown . B - H , Representative images of the SCN staining in Col , and the indicated single , double , and triple mutant roots . QC positions are marked by yellow arrowhead . I , Analyses of the SCN staining for CSC phenotypes . Frequencies of roots showing 0 , 1 , 2 , or 3 CSC layers are plotted as bar graphs . J , Schematic representation of a transversal section of an Arabidopsis RM . QC cells are marked in red , CEI initials are marked in turquoise . K - Q , Representative images of transversal sections with QC cells outlined in yellow . R , Analyses of the SCN staining for QC division phenotypes . Frequencies of roots showing 0 , 1 , 2 , 3 or ≥ 4 dividing QC cells are plotted as bar graphs . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype and results from 2 - 5 technical replicates per genotype . QC = quiescent center , CSC = columella stem cell , CEI = cortex endodermis initial , SCN = stem cell niche , mPSPI = modified pseudo - Schiff propidium iodide , EdU = 5 - ethynyl - 2 ' - deoxyuridine , scale bars represent 5 µm .

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A, Schematic representation of a longitudinal section of an Arabidopsis RM. QC cells are marked in red, CSCs are marked in dark blue, CCs in light blue. Combined mPSPI (grey) and EdU (red) staining for 24 hours (SCN staining) to analyse the CSC (A-I) and QC division phenotype (J-R) within the same roots are shown. B-H, Representative images of the SCN staining in Col, and the indicated single, double, and triple mutant roots. QC positions are marked by yellow arrowhead. I, Analyses of the SCN staining for CSC phenotypes. Frequencies of roots showing 0, 1, 2, or 3 CSC layers are plotted as bar graphs. J, Schematic representation of a transversal section of an Arabidopsis RM. QC cells are marked in red, CEI initials are marked in turquoise. K-Q, Representative images of transversal sections with QC cells outlined in yellow. R, Analyses of the SCN staining for QC division phenotypes. Frequencies of roots showing 0, 1, 2, 3 or ≥4 dividing QC cells are plotted as bar graphs. Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from 2-5 technical replicates per genotype. QC = quiescent center, CSC = columella stem cell, CEI = cortex endodermis initial, SCN = stem cell niche, mPSPI = modified pseudo-Schiff propidium iodide, EdU = 5-ethynyl-2'-deoxyuridine, scale bars represent 5 µm.


The combined results of the SCN staining 3 are shown as 2D plots to visualise the correlation of the CSC layer and QC division phenotypes . Number of CSC layers are shown on the y axis and the QC division phenotype is shown on the x axis . The darker the colour , the more roots show the respective phenotype ( see colour gradient top right indicating the frequencies in percent ) . Col wild - type roots show one layer of CSCs and no EdU stained cells ( no QC division ) after 24 h EdU staining . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype and results from 2 - 5 technical replicates per genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 combined results of the SCN staining 3 are shown as 2D plots to visualise the correlation of the CSC layer and QC division phenotypes. Number of CSC layers are shown on the y axis and the QC division phenotype is shown on the x axis. The darker the colour, the more roots show the respective phenotype (see colour gradient top right indicating the frequencies in percent). Col wild-type roots show one layer of CSCs and no EdU stained cells (no QC division) after 24 h EdU staining. Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from 2-5 technical replicates per genotype.


A - H , Fluorescence Lifetime Imaging ( FLIM ) results of transiently expressing N . benthamiana leaf epidermal cells . A - H , Representative FLIM images of WOX5 - mVenus ( donor only ) plus the indicated acceptors after a pixel - wise mono - exponential fit of the mVenus fluorescence signal . The fluorescence lifetime of WOX5 - mVenus in ns is color - coded . Low lifetimes ( blue ) due to FRET indicate strong interaction of the two proteins and high lifetimes ( red ) indicate weaker or no interaction . Scale bars represent 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H, Fluorescence Lifetime Imaging (FLIM) results of transiently expressing N. benthamiana leaf epidermal cells. A-H, Representative FLIM images of WOX5-mVenus (donor only) plus the indicated acceptors after a pixel-wise mono-exponential fit of the mVenus fluorescence signal. The fluorescence lifetime of WOX5-mVenus in ns is color-coded. Low lifetimes (blue) due to FRET indicate strong interaction of the two proteins and high lifetimes (red) indicate weaker or no interaction. Scale bars represent 5 µm.


I , Fluorescence lifetimes in ns are summarized in combined jitter and box plots . The dashed line represents the fluorescence lifetime mean value of the WOX5 - mV co - expressed with free mCh as negative control . The one - way ANOVA and Holm - Sidak post - hoc multiple comparisons test was used to test for statistical significance . Samples with identical letters do not show significant differences ( α = 0 . 01 ) . Number of nuclei analyzed ( n ) ( biological replicates ) is indicated and results from 2 to 9 technical replicates . Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , ▪ = mean value .

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

I, Fluorescence lifetimes in ns are summarized in combined jitter and box plots. The dashed line represents the fluorescence lifetime mean value of the WOX5-mV co-expressed with free mCh as negative control. The one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test was used to test for statistical significance. Samples with identical letters do not show significant differences (α = 0.01). Number of nuclei analyzed (n) (biological replicates) is indicated and results from 2 to 9 technical replicates. Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, ▪ = mean value.


J , Representative FLIM images of WOX5 - mVenus ( donor only ) after a pixel - wise mono - exponential fit of the mVenus fluorescence signal . The fluorescence lifetime of WOX5 - mVenus in ns is color - coded . Low lifetimes ( blue ) due to FRET indicate strong interaction of the two proteins and high lifetimes ( red ) indicate weaker or no interaction . Scale bars represent 5 µm .

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

J, Representative FLIM images of WOX5-mVenus (donor only) after a pixel-wise mono-exponential fit of the mVenus fluorescence signal. The fluorescence lifetime of WOX5-mVenus in ns is color-coded. Low lifetimes (blue) due to FRET indicate strong interaction of the two proteins and high lifetimes (red) indicate weaker or no interaction. Scale bars represent 5 µm.


K , Individual nuclei showing nuclear bodies during co - expression of WOX5 - mV and PLT3 - mCh were analysed for WOX5 - mV lifetime in the nuclear bodies or nucleoplasm separately ( n = 7 ( biological replicates ) , from four technical replicates . mCh = mCherry . mV = mVenus .

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K, Individual nuclei showing nuclear bodies during co-expression of WOX5-mV and PLT3-mCh were analysed for WOX5-mV lifetime in the nuclear bodies or nucleoplasm separately (n = 7 (biological replicates), from four technical replicates. mCh = mCherry. mV = mVenus.


( A ) Masson ' s trichrome representative images of pulmonary fibrosis and quantification .

(A) Masson's trichrome representative images of pulmonary fibrosis and quantification.


( B ) Relative transcript expression of Fibronectin , ( C ) Collagen I and ( D ) Collagen III to GAPDH .

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(B) Relative transcript expression of Fibronectin, (C) Collagen I and (D) Collagen III to GAPDH.


( E ) Masson ' s trichrome representative images of vascular wall thickening and quantification .

(E) Masson's trichrome representative images of vascular wall thickening and quantification.


( F ) Masson ' s trichrome representative images and correlation between lung fibrosis and vascular wall thickness in PF and PF - PH patients . Out of the 14 PF and PF - PH patients , 3 of them had no non - fibrotic area and therefore were excluded for comparison between non - fibrotic and fibrotic areas ( n = 11 / group ; Ashcroft score non - fibrosis : 0 - 2 ; mild - fibrosis : 3 - 5 ; severe - fibrosis > 5 ) ( * compared to non - fibrotic areas of the same group of patients ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Masson's trichrome representative images and correlation between lung fibrosis and vascular wall thickness in PF and PF-PH patients. Out of the 14 PF and PF-PH patients, 3 of them had no non-fibrotic area and therefore were excluded for comparison between non-fibrotic and fibrotic areas (n=11/group; Ashcroft score non-fibrosis: 0-2; mild-fibrosis: 3-5; severe-fibrosis >5) (* compared to non-fibrotic areas of the same group of patients).


( G ) Masson ' s trichrome representative images of vascular wall thickening in non - fibrotic area ( Ashcroft score 0 - 2 ) and fibrotic area ( Ashcroft score > 2 ) and quantification in PF and PF - PH . The number of samples per group for each experiment are written within each bar graph .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Masson's trichrome representative images of vascular wall thickening in non-fibrotic area (Ashcroft score 0-2) and fibrotic area (Ashcroft score >2) and quantification in PF and PF-PH. The number of samples per group for each experiment are written within each bar graph.


( A ) Representative images of Ki67 - positive pulmonary vascular endothelial cells ( EC , vWF in green ) and smooth muscle cells ( SMC , aSMA in white ) and quantification ( * vs PF , * p < 0 . 05 , * * * p < 0 . 001 ) .

(A) Representative images of Ki67-positive pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white) and quantification (* vs PF, *p<0.05, ***p<0.001).


Relative expression of Slug mRNA ( B ) normalized to GAPDH in PF and PF - PH patients .

Relative expression of Slug mRNA (B) normalized to GAPDH in PF and PF-PH patients.


Relative expression of Slug protein ( C ) normalized to GAPDH in PF and PF - PH patients .

Relative expression of Slug protein (C) normalized to GAPDH in PF and PF-PH patients.


( D ) Representative images of Slug ( red ) expression in bronchial epithelial cells ( EpC ) , fibroblasts ( Fib ) ( vimentin in green ) macrophages ( Mф , CD68 in green ) pulmonary vascular endothelial cells ( EC , von Willebrand Factor in green ) and pulmonary vascular smooth muscle cells ( SMC , aSMA in white ) ; and quantification .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib) (vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, von Willebrand Factor in green) and pulmonary vascular smooth muscle cells (SMC, aSMA in white); and quantification.


( E ) Relative CD68 mRNA expression normalized to

(E) Relative CD68 mRNA expression normalized to GAPDH


( F ) Representative images of Slug ( red ) expression in macrophages ( Mф , CD68 in green ) in non - fibrotic and fibrotic areas of the lung in PF and PF - PH patients and quantification . The number of samples per group for each experiment are included within each bar graph .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in PF and PF-PH patients and quantification. The number of samples per group for each experiment are included within each bar graph.


( A ) Heatmap of the 20 genes up - regulated in GSE24988 microarray and known to be Slug transcriptionnal target .

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(A) Heatmap of the 20 genes up-regulated in GSE24988 microarray and known to be Slug transcriptionnal target.


( C ) Heatmap of the seven transcriptional targets of Slug known to promote proliferation and to be extracellular .

(C) Heatmap of the seven transcriptional targets of Slug known to promote proliferation and to be extracellular.


Relative expression of Prolactin - induced protein ( PIP ) mRNA ( D ) normalized to GAPDH in PF and PF - PH patients .

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Relative expression of Prolactin-induced protein (PIP) mRNA (D) normalized to GAPDH in PF and PF-PH patients.


Relative expression of Prolactin - induced protein ( PIP ) expression normalized to GAPDH in PF and PF - PH patients .

Relative expression of Prolactin-induced protein (PIP) expression normalized to GAPDH in PF and PF-PH patients.


( F ) Representative images of PIP immunohistochemistry showing its extracellular localization and quantification .

(F) Representative images of PIP immunohistochemistry showing its extracellular localization and quantification.


Proliferation assay on healthy fibroblast ( G ) Pulmonary arterial EC ( H ) and on healthy pulmonary arterial SMC ( I in the presence or absence of PIP protein . The number of samples per group for each experiment are included within each bar graph .

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Proliferation assay on healthy fibroblast (G) Pulmonary arterial EC (H) and on healthy pulmonary arterial SMC (I in the presence or absence of PIP protein. The number of samples per group for each experiment are included within each bar graph.


Proliferation assay on healthy pulmonary arterial SMC J ) in the presence or absence of PIP protein . The number of samples per group for each experiment are included within each bar graph .

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Proliferation assay on healthy pulmonary arterial SMC J) in the presence or absence of PIP protein. The number of samples per group for each experiment are included within each bar graph.


( A ) Masson ' s trichrome representative images of lung fibrosis and quantification two weeks after PBS or bleomycin instillation .

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(A) Masson's trichrome representative images of lung fibrosis and quantification two weeks after PBS or bleomycin instillation.


Quantification of ( B ) fibronectin , ( C ) Collagen I and ( D ) Collagen III mRNA .

Quantification of (B) fibronectin, (C) Collagen I and (D) Collagen III mRNA.


( F ) Right ventricular systolic pressure ( RVSP ) and ( G ) Right ventricular hypertrophy index following the experimental protocol .

(F) Right ventricular systolic pressure (RVSP) and (G) Right ventricular hypertrophy index following the experimental protocol.


( H ) Masson ' s trichrome representative images and quantification of Ashcroft score in the experimental model .

(H) Masson's trichrome representative images and quantification of Ashcroft score in the experimental model.


Relative expression of fibronectin ( I ) , collagen I ( J ) and collagen III ( K ) mRNA normalized to GAPDH .

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Relative expression of fibronectin (I), collagen I (J) and collagen III (K) mRNA normalized to GAPDH.


( L ) Masson ' s trichrome representative images and quantification of vascular remodeling .

(L) Masson's trichrome representative images and quantification of vascular remodeling.


( M ) Masson ' s trichrome representative images and quantification of the vascular remodeling between fibrotic and non - fibrotic areas in the experimental model . The number of samples per group for each experiment are included within each bar graph .

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(M) Masson's trichrome representative images and quantification of the vascular remodeling between fibrotic and non-fibrotic areas in the experimental model. The number of samples per group for each experiment are included within each bar graph.


( A ) Representative images and quantification of Ki67 - positive ( red ) pulmonary vascular endothelial cells ( EC , vWF in green ) and smooth muscle cells ( SMC ) in the experimental model .

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(A) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC) in the experimental model.


Relative expression of Slug mRNA ( B ) normalized to GAPDH .

Relative expression of Slug mRNA (B) normalized to GAPDH.


Relative expression of Slug protein ( C ) normalized to GAPDH .

Relative expression of Slug protein (C) normalized to GAPDH.


( D ) Representative images of Slug ( red ) expression in bronchial epithelial cells ( EpC ) , fibroblasts ( Fib , vimentin in green ) macrophages ( Mф , CD68 in green ) pulmonary vascular endothelial cells ( EC , vWF ) and smooth muscle cells ( SMC , aSMA in white ) ; and quantification . ( * vs Bleo , p < 0 . 05 )

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

(D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib, vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, vWF) and smooth muscle cells (SMC, aSMA in white); and quantification. (* vs Bleo, p<0.05)


( E ) CD68 mRNA quantification in Bleo and Bleo - MCT

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(E) CD68 mRNA quantification in Bleo and Bleo-MCT rats


( F ) Representative images of Slug ( red ) expression in macrophages ( Mф , CD68 in green ) in non - fibrotic and fibrotic areas of the lung in in Bleo and Bleo - MCT rats and quantification . The number of samples per group for each experiment are included within each bar graph .

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(F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in in Bleo and Bleo-MCT rats and quantification. The number of samples per group for each experiment are included within each bar graph.


Relative expression of PIP mRNA ( A ) normalized to GAPDH .

Relative expression of PIP mRNA (A) normalized to GAPDH.


Relative expression of PIP protein ( B ) normalized to GAPDH .

Relative expression of PIP protein (B) normalized to GAPDH.


( C ) Representative images and PIP quantification . The number of samples per group for each experiment are included within each bar graph .

(C) Representative images and PIP quantification. The number of samples per group for each experiment are included within each bar graph.


Relative expression of Slug protein ( B ) normalized to GAPDH .

Relative expression of Slug protein (B) normalized to GAPDH.


Relative expression of Slug mRNA ( C ) normalized to GAPDH .

Relative expression of Slug mRNA (C) normalized to GAPDH.


( D ) right ventricular systolic pressure ( RVSP ) and ( E ) right ventricular hypertrophy index in PF - PH rats treated with Si - Slug compared to Si - Scrm .

(D) right ventricular systolic pressure (RVSP) and (E) right ventricular hypertrophy index in PF-PH rats treated with Si-Slug compared to Si-Scrm.


Relative expression of fibronectin ( F ) , Collagen I ( G ) and Collagen III ( H ) normalized to GAPDH .

Relative expression of fibronectin (F), Collagen I (G) and Collagen III (H) normalized to GAPDH.


( I ) Masson ' s trichrome representative images and quantification of the Ashcroft score . ( J ) Masson ' s trichrome representative images and quantification of vascular wall thickness .

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(I) Masson's trichrome representative images and quantification of the Ashcroft score. (J) Masson's trichrome representative images and quantification of vascular wall thickness.


( K ) Masson ' s trichrome representative images and quantification of the vascular remodeling between non - fibrotic and fibrotic areas of the lung .

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(K) Masson's trichrome representative images and quantification of the vascular remodeling between non-fibrotic and fibrotic areas of the lung.


( L ) Representative images and quantification of Ki67 - positive ( red ) pulmonary vascular endothelial cells ( EC , vWF in green ) and smooth muscle cells ( SMC , aSMA in white ) . The number of samples per group for each experiment are included within each bar graph .

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

(L) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white). The number of samples per group for each experiment are included within each bar graph.


( A ) Representative images and quantification of Slug - positive macrophages ( slug , red ; CD68 green ) .

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(A) Representative images and quantification of Slug-positive macrophages (slug, red; CD68 green).


( B ) Relative expression of CD68 mRNA normalized to GAPDH .

(B) Relative expression of CD68 mRNA normalized to GAPDH.


( C ) Representative images and quantification of Slug - positive macrophages between non - fibrotic and fibrotic areas of the lung .

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(C) Representative images and quantification of Slug-positive macrophages between non-fibrotic and fibrotic areas of the lung.


Relative expression of PIP mRNA ( D ) normalized to GAPDH .

Relative expression of PIP mRNA (D) normalized to GAPDH.


Relative expression of PIP protein ( E ) normalized to GAPDH .

Relative expression of PIP protein (E) normalized to GAPDH.


( F ) Representative images and quantification of PIP immunohistochemistry .

(F) Representative images and quantification of PIP immunohistochemistry.


B Immunoblot showing CDK5RAP2 levels in Cdk5rap2 wild - type ( WT ) , heterozygous ( HET ) and null erythroid progenitors ( EPs ) isolated from fetal livers . Actin was used as loading control . * * indicates nonspecific band . C Quantification of mean protein levels from ( B ) . Numbers in brackets correspond to number of embryos analyzed . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of embryos ( C , Statistical significances were determined by One - way ANOVA test with Tukey ' s ( C , test * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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B Immunoblot showing CDK5RAP2 levels in Cdk5rap2 wild-type (WT), heterozygous (HET) and null erythroid progenitors (EPs) isolated from fetal livers. Actin was used as loading control. ** indicates nonspecific band. C Quantification of mean protein levels from (B). Numbers in brackets correspond to number of embryos analyzed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos (C, Statistical significances were determined by One-way ANOVA test with Tukey's (C, test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


D Immunofluorescence images of Cdk5rap2 WT , HET and null erythroid progenitors isolated from fetal livers . Progenitors were stained for CDK5RAP2 ( grey ) , γ - tubulin ( magenta ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 3 μm . Insets show higher magnification of centrosomes . Scale bar , 1 μm . E Quantification of mean centrosomal signal intensities of CDK5RAP2 and γ - tubulin from ( D ) . Numbers in brackets correspond to number of embryos analyzed with a total number of 470 ( WT ) , 406 ( HET ) and 379 ( null ) progenitors . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of embryos E , Statistical significances were determined by One - way ANOVA test with Tukey ' s E , test * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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D Immunofluorescence images of Cdk5rap2 WT, HET and null erythroid progenitors isolated from fetal livers. Progenitors were stained for CDK5RAP2 (grey), γ-tubulin (magenta) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 3 μm. Insets show higher magnification of centrosomes. Scale bar, 1 μm. E Quantification of mean centrosomal signal intensities of CDK5RAP2 and γ-tubulin from (D). Numbers in brackets correspond to number of embryos analyzed with a total number of 470 (WT), 406 (HET) and 379 (null) progenitors. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos E, Statistical significances were determined by One-way ANOVA test with Tukey's E, test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


I ImageStream images of ex vivo cultured enucleating EBs and reticulocytes . Cells were stained for TER119 ( erythroid marker , green ) and DNA ( Hoechst , blue ) . BF : brightfield . Scale bar , 5 μm . J Quantification of enucleating EBs and reticulocytes after 48 hours ( T48 ) in ex vivo culture . Genotypes are as indicated . The numbers in brackets correspond to the number of embryos analyzed . K Quantification of enucleating EB size from ( I ) . The numbers in brackets refer to the number of embryos analyzed . Data information : Box plots show 5th , 95th ( whiskers ) and 25th , 50th and 75th percentile ( boxes ) . Statistical analysis was based on the number of embryos I and J ) Statistical significances were determined by One - way ANOVA test with Tukey ' s J ) or Kruskal Wallis test with Dunn ' s ( K ) multiple comparisons . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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I ImageStream images of ex vivo cultured enucleating EBs and reticulocytes. Cells were stained for TER119 (erythroid marker, green) and DNA (Hoechst, blue). BF: brightfield. Scale bar, 5 μm. J Quantification of enucleating EBs and reticulocytes after 48 hours (T48) in ex vivo culture. Genotypes are as indicated. The numbers in brackets correspond to the number of embryos analyzed. K Quantification of enucleating EB size from (I). The numbers in brackets refer to the number of embryos analyzed. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Statistical analysis was based on the number of embryos I and J) Statistical significances were determined by One-way ANOVA test with Tukey's J) or Kruskal Wallis test with Dunn's (K) multiple comparisons.*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


A Deconvolved STED images of centrosomes in ex vivo cultured wild - type non - enucleating EBs ( non - enucl . ) or enucleating EBs / reticulocytes ( enucl . / RetiC ) . Cells were stained for γ - tubulin ( magenta ) and centriolar ( CP110 , CEP135 ) or PCM ( CDK5RAP2 , PCNT ) proteins ( cyan ) . Scale bar , 400 nm .

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A Deconvolved STED images of centrosomes in ex vivo cultured wild-type non-enucleating EBs (non-enucl.) or enucleating EBs/reticulocytes (enucl./RetiC). Cells were stained for γ-tubulin (magenta) and centriolar (CP110, CEP135) or PCM (CDK5RAP2, PCNT) proteins (cyan). Scale bar, 400 nm.


E ImageStream images showing EBs enucleating with 2N or 4N DNA content at different stages from ( B ) . EBs were stained for DNA ( Hoechst , blue ) and TER119 ( erythroid marker , green ) . Scale bar , 5 μm . BF : brightfield . F Quantification of enucleation stages according to DNA content from ( B ) . 4 ( WT ) , 14 ( HET ) and 8 ( null ) embryos were analyzed . Data information : Bar graphs display mean ± s . d . All statistical analysis were based on the number of embryos . Statistical significance was determined by Two - way ANOVA with Tukey ' s ( F ) multiple comparisons . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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E ImageStream images showing EBs enucleating with 2N or 4N DNA content at different stages from (B). EBs were stained for DNA (Hoechst, blue) and TER119 (erythroid marker, green). Scale bar, 5 μm. BF: brightfield. F Quantification of enucleation stages according to DNA content from (B). 4 (WT), 14 (HET) and 8 (null) embryos were analyzed. Data information: Bar graphs display mean ± s.d. All statistical analysis were based on the number of embryos. Statistical significance was determined by Two-way ANOVA with Tukey's (F) multiple comparisons. **P≤0.01, ***P≤0.001, ****P≤0.0001.


B Immunofluorescence images of untreated ( untx ) or CB - treated erythroid progenitors at stated differentiation stages after 24 ( T24 ) and 48 ( T48 ) hours in ex vivo culture . Cells were stained for γ - tubulin ( magenta ) , TER119 ( erythroid marker , green ) and DNA ( Hoechst , blue ) . Scale bar , 3 μm .

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B Immunofluorescence images of untreated (untx) or CB-treated erythroid progenitors at stated differentiation stages after 24 (T24) and 48 (T48) hours in ex vivo culture. Cells were stained for γ-tubulin (magenta), TER119 (erythroid marker, green) and DNA (Hoechst, blue). Scale bar, 3 μm.


C Quantification of centrosome numbers ( marked by γ - tubulin ) upon CB treatment from ( B ) . For the left panel , in total 632 ( untx , T24 ) , 637 ( CB , T24 ) , 544 ( untx , T48 ) and 467 ( CB , T48 ) cells from two litters were analyzed . T24 = 24 hours . T48 = 48 hours . For the right panel , in total 799 ( untx , non - enucl ) , 142 ( untx , enucl ) , 235 ( untx , RetiC ) , 839 ( CB , non - enucl ) , 127 ( CB , enucl ) and 131 ( CB , RetiC ) cells from two litters were analysed . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of litters ( C * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

C Quantification of centrosome numbers (marked by γ-tubulin) upon CB treatment from (B). For the left panel, in total 632 (untx, T24), 637 (CB, T24), 544 (untx, T48) and 467 (CB, T48) cells from two litters were analyzed. T24 = 24 hours. T48 = 48 hours. For the right panel, in total 799 (untx, non-enucl), 142 (untx, enucl), 235 (untx, RetiC), 839 (CB, non-enucl), 127 (CB, enucl) and 131 (CB, RetiC) cells from two litters were analysed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (C *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


A Immunofluorescence images of untreated ( untx ) or centrinone - B ( CB ) - treated cells at 36 hours ( T36 ) of ex vivo culture . Representative examples for different mitotic spindle morphologies are shown . Cells were stained for α - tubulin ( magenta ) , γ - tubulin ( grey ) , phospho - Histone H3 ( pHH3 , green ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 2 μm . B Quantification of mitotic spindle morphologies in untreated ( untx ) or centrinone - B ( CB ) - treated cells at T24 or T36 of ex vivo culture . Graph depicts percentage of spindle phenotypes . In total 315 ( untx , T24 ) , 367 ( CB , T24 ) , 295 ( untx , T36 ) , 282 ( CB , T36 ) cells were analyzed from 3 litters . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of litters ( B ) Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons test ( B , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

A Immunofluorescence images of untreated (untx) or centrinone-B (CB)-treated cells at 36 hours (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained for α-tubulin (magenta), γ-tubulin (grey), phospho-Histone H3 (pHH3, green) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. B Quantification of mitotic spindle morphologies in untreated (untx) or centrinone-B (CB)-treated cells at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle phenotypes. In total 315 (untx, T24), 367 (CB, T24), 295 (untx, T36), 282 (CB, T36) cells were analyzed from 3 litters. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (B) Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test (B, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


C Immunofluorescence images of Cdk5rap2HET and Cdk5rap2null cells at 36 hours ( T36 ) of ex vivo culture . Representative examples for different mitotic spindle morphologies are shown . Cells were stained for α - tubulin ( magenta ) , γ - tubulin ( grey ) , pHH3 ( green ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 2 μm . D Quantification of mitotic spindle morphology at T24 or T36 of ex vivo culture . Graph depicts percentage of spindle phenotypes . At T24 , 3 Cdk5rap2WT ( 323 cells ) , 3 Cdk5rap2HET ( 296 cells ) and 2 Cdk5rap2null ( 197 cells ) embryos were analyzed . At T36 , 5 Cdk5rap2WT ( 387 cells ) , 6 Cdk5rap2HET ( 384 cells ) and 3 Cdk5rap2null ( 202 cells ) embryos were analyzed . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of embryos ( D , Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons test D , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

C Immunofluorescence images of Cdk5rap2HET and Cdk5rap2null cells at 36 hours (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained for α-tubulin (magenta), γ-tubulin (grey), pHH3 (green) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. D Quantification of mitotic spindle morphology at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle phenotypes. At T24, 3 Cdk5rap2WT (323 cells), 3 Cdk5rap2HET (296 cells) and 2 Cdk5rap2null (197 cells) embryos were analyzed. At T36, 5 Cdk5rap2WT (387 cells), 6 Cdk5rap2HET (384 cells) and 3 Cdk5rap2null (202 cells) embryos were analyzed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos (D, Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test D, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


E Schematic ( left ) shows experimental outline for nocodazole treatment of ex vivo cultured erythroid progenitors at indicated time points . Representative ImageStream images of ex vivo cultured cells at different mitotic stages ( right ) . Cells were stained for phospho - Histone H3 ( pHH3 , green ) , TER119 ( erythroid marker , magenta ) and DNA ( Hoechst , blue ) . BF : brightfield . Scale bar , 10 μm . F and G Quantification of pHH3pos , TER119pos cells and mitotic stages ( see text for details ) after 36 hours ( T36 ) upon DMSO or nocodazole treatment according to ( E ) using ImageStream . Number of embryos analyzed is shown in brackets . Data information : Box plots show 5th , 95th ( whiskers ) and 25th , 50th and 75th percentile ( boxes ) . Statistical analysis was based on the number of embryos F and G ) . Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons test F and G ) . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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 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 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

E Schematic (left) shows experimental outline for nocodazole treatment of ex vivo cultured erythroid progenitors at indicated time points. Representative ImageStream images of ex vivo cultured cells at different mitotic stages (right). Cells were stained for phospho-Histone H3 (pHH3, green), TER119 (erythroid marker, magenta) and DNA (Hoechst, blue). BF: brightfield. Scale bar, 10 μm. F and G Quantification of pHH3pos, TER119pos cells and mitotic stages (see text for details) after 36 hours (T36) upon DMSO or nocodazole treatment according to (E) using ImageStream. Number of embryos analyzed is shown in brackets. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Statistical analysis was based on the number of embryos F and G). Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test F and G).*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


B Immunofluorescence images of cells in interphase ( I ) and mitosis ( M ) at 24 ( T24 ) and 36 ( T36 ) hours of ex vivo culture . Cells were stained for γ - tubulin ( magenta ) , protein of interest ( POI , CDK5RAP2 , PCNT or CEP192 in grey ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 2 μm . C Quantification of mean centrosomal signal intensities of PCM proteins from ( B ) . Three litters were analyzed with the following number of cells for I , T24 ; M , T24 ; I , T36 and M , T36 : 290 , 157 , 259 and 174 ( CDK5RAP2 ) ; 291 , 190 , 203 and 181 ( PCNT ) ; 314 , 122 , 283 and 152 ( CEP192 ) ; 895 , 465 , 720 and 493 ( γ - tubulin ) . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of litters ( C Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons test * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

B Immunofluorescence images of cells in interphase (I) and mitosis (M) at 24 (T24) and 36 (T36) hours of ex vivo culture. Cells were stained for γ-tubulin (magenta), protein of interest (POI, CDK5RAP2, PCNT or CEP192 in grey) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. C Quantification of mean centrosomal signal intensities of PCM proteins from (B). Three litters were analyzed with the following number of cells for I, T24; M, T24; I, T36 and M, T36: 290, 157, 259 and 174 (CDK5RAP2); 291, 190, 203 and 181 (PCNT); 314, 122, 283 and 152 (CEP192); 895, 465, 720 and 493 (γ-tubulin). Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (C Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


A Immunoblot analysis of TP53 , P27 and P21 levels in ex vivo cultured Cdk5rap2 wild - type ( WT ) , heterozygous ( HET ) and null EBs after 24 ( T24 ) and 48 ( T48 ) hours . GAPDH was used as loading control . * * indicates unspecific band . B Quantification of mean protein levels from ( A ) . Numbers in brackets represent number of embryos analyzed . Data information : Box plots show 5th , 95th ( whiskers ) and 25th , 50th and 75th percentile ( boxes ) . All statistical analysis were based on number of embryos . All statistical significances were determined by One - way ANOVA with Tukey ' s multiple comparisons test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblot analysis of TP53, P27 and P21 levels in ex vivo cultured Cdk5rap2 wild-type (WT), heterozygous (HET) and null EBs after 24 (T24) and 48 (T48) hours. GAPDH was used as loading control. ** indicates unspecific band. B Quantification of mean protein levels from (A). Numbers in brackets represent number of embryos analyzed. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). All statistical analysis were based on number of embryos. All statistical significances were determined by One-way ANOVA with Tukey's multiple comparisons test.*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


B . Analysis of cell cycle arrest ( in G2 ) in response to DSB induction assayed by flow cytometry .

B. Analysis of cell cycle arrest (in G2) in response to DSB induction assayed by flow cytometry.


C . Rad53 phosphorylation ( Rad53 - P ) in response to DSB assayed by western blotting .

C. Rad53 phosphorylation (Rad53-P) in response to DSB assayed by western blotting.


D . Cell survival upon DSB induction . Average ± SD ( n = 4 ) is shown for each genotype .

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

D. Cell survival upon DSB induction. Average ± SD (n=4) is shown for each genotype.


O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Srs2 requirement in de novo telomere addition in cells with and without functional HR. All strains are pif1-m2. Strains used: NK1264; NK2375, NK2376; NK2014, NK2015; NK2451, NK2452; NK2012, NK2013; NK2457, NK2458; NK2363, NK2364; NK2469; 2369, NK2370; NK2473-2475.


D . Dynamics of ( TG1 - 3 ) n addition monitored by qPCR through a time - course experiment ( asynchronous populations ) . The Y axis shows a fold increase in de novo telomere - specific PCR product relative to the background levels at 0 h and normalised against an internal control ( ARO1 locus ) . Average ± SD ( n = 3 ) is shown for each time - point of each genotype . Strains used : NK3292 , NK3293 ; NK4670 , NK4671 ; NK4112 , NK4113 ; NK4114 , NK4115 ; NK3292 est2 ∆ , NK3293 est2 ∆ ; NK4232 , NK4233 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Dynamics of (TG1-3)n addition monitored by qPCR through a time-course experiment (asynchronous populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Strains used: NK3292, NK3293; NK4670, NK4671; NK4112, NK4113; NK4114, NK4115; NK3292 est2∆, NK3293 est2∆; NK4232, NK4233.


F . Comparative analysis of ssDNA / dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time - course experiment ( synchronised populations ) . The Y axis shows a fold increase in de novo telomere - specific PCR product relative to the background levels at 0 h and normalised against an internal control ( ARO1 locus ) . Average ± SD ( n = 3 ) is shown for each time - point of each genotype . Top set of error bars represents SD in relative increase of the de novo telomere - specific PCR product ( as in panel D ) while the lower set of error bars corresponds to quantifications of ss / dsDNA fractions . Strains used : NK3292 , NK3293 ; NK4670 , NK4671 .

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

F. Comparative analysis of ssDNA/dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time-course experiment (synchronised populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Top set of error bars represents SD in relative increase of the de novo telomere-specific PCR product (as in panel D) while the lower set of error bars corresponds to quantifications of ss/dsDNA fractions. Strains used: NK3292, NK3293; NK4670, NK4671.


B . Southern blot analysis of re - synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C . DNA was digested with EcoRI and BamHI , resolved on 0 . 7 % agarose gels , transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes ( three RS probes and a reference probe , REF , hybridizing to an ARS522 - containing fragment on chr . V which is not involved in the repair ) . A representative image of one of the three repeats is shown . C . Re - synthesis of resected DNA on chr . VIIL in SRS2 and srs2Δ cells ( solid and dashed lines , respectively ) at the distance of 15 . 2 ( RS15 . 2 ) , 6 . 8 ( RS6 . 8 ) and 2 . 6 ( RS2 . 6 ) kb away from the homology region . Average ± SD ( n = 3 ) is shown for each time point .

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

B. Southern blot analysis of re-synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three RS probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown.C. Re-synthesis of resected DNA on chr.VIIL in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 15.2 (RS15.2), 6.8 (RS6.8) and 2.6 (RS2.6) kb away from the homology region. Average ± SD (n=3) is shown for each time point.


D . Southern blot analysis of BIR - dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D . DNA was digested with EcoRI and BamHI , resolved on 0 . 7 % agarose gels , transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes ( three BIR probes and a reference probe , REF , hybridizing to an ARS522 - containing fragment on chr . V which is not involved in the repair ) . A representative image of one of the three repeats is shown . C - control strain NK3980 . E . BIR - dependent DNA synthesis in SRS2 and srs2Δ cells ( solid and dashed lines , respectively ) at the distance of 6 ( BIR6 ) , 36 ( BIR36 ) and 77 ( BIR77 ) kb away from the homology region . Average ± SD ( n = 3 ) is shown for each time point .

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 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 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

D. Southern blot analysis of BIR-dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three BIR probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown. C - control strain NK3980.E. BIR-dependent DNA synthesis in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 6 (BIR6), 36 (BIR36) and 77 (BIR77) kb away from the homology region. Average ± SD (n=3) is shown for each time point.


B . Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in panel A . Average ± SD ( n = 4 ) is shown for each genotype . Strains used in B and D : NK4691 - 4693 ; NK4805 - 4808 ; NK5081 - 5084 ; NK5085 - 5091 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in panel A. Average ± SD (n=4) is shown for each genotype. Strains used in B and D: NK4691-4693; NK4805-4808; NK5081-5084; NK5085-5091.


D . Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51 . Also see Figure EV3 for blot images . Average ± SD ( n = 4 ) is shown for each time point .

O O O O O O O O O O O O O O O O O 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. Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51. Also see Figure EV3 for blot images. Average ± SD (n=4) is shown for each time point.


A , B : serum levels of S1P ( A ) and apoM ( B ) in HLT ( n = 47 ) and COV ( n = 111 ) patients . The box - plots represent the interquartile range with median value ( central line ) ; the whiskers represent the measured range of HLT and COV . Each measurement was run in triplicate , and performed at least twice . Two - tailed Student ' s t - test was used for statistical analysis . * * * * p < 0 . 0001 .

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

A, B: serum levels of S1P (A) and apoM (B) in HLT (n=47) and COV (n=111) patients. The box-plots represent the interquartile range with median value (central line); the whiskers represent the measured range of HLT and COV. Each measurement was run in triplicate, and performed at least twice. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.


Pearson correlation between S1P and apoM ( C ) Scatter plots , together with the fitted regression line , are shown . Pearson correlation was performed for statistical analysis . 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

Pearson correlation between S1P and apoM (C) Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.


Pearson correlation between S1P and RBC number ( D ) , HGB concentration ( E ) , HCT value ( F ) . Scatter plots , together with the fitted regression line , are shown . Pearson correlation was performed for statistical analysis . Exact p values or * * * * p < 0 . 0001 are reported .

O O O B-MEASURED_VAR 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

Pearson correlation between S1P and RBC number (D), HGB concentration (E), HCT value (F). Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.


A , B : correlations between S1P and HDL - C ( A ) , and albumin ( B ) . Data information : Scatter plots and fitted regression line are shown in each figure . Each S1P measurement was run in triplicate , and performed at least twice in independent assays . Pearson correlation was performed for statistical analysis . Exact p values or * * * * p < 0 . 0001 are reported in the figure .

O O O O O O B-MEASURED_VAR 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 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, B: correlations between S1P and HDL-C (A), and albumin (B). Data information: Scatter plots and fitted regression line are shown in each figure. Each S1P measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.


C , D : correlations between apoM and HDL - C ( C ) and albumin ( D ) . Data information : Scatter plots and fitted regression line are shown in each figure . Each apoM measurement was run in triplicate , and performed at least twice in independent assays . Pearson correlation was performed for statistical analysis . Exact p values or * * * * p < 0 . 0001 are reported in the figure .

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

C, D: correlations between apoM and HDL-C (C) and albumin (D). Data information: Scatter plots and fitted regression line are shown in each figure. Each apoM measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.