e , TβRII + crEVs ( left panel ) , USP8 enzyme activity ( midde panel ) USP8 mRNA ( right panel ) from breast cancer with or without clinical response to combined therapy using anti - PD - L1 ( Atezolizumab ) paclitaxel . R , responders ; n = 10 ( left panel ) , n = 5 ( middle right panels , only 5 - derived tissue samples obtained ) . NR , non - responders ; n = 10 ( left panel ) , n = 6 ( middle right panels , only 5 - 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 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 stably ovexpressed with USP8 - WT ( n = 7 ) or USP8 - CS ( n = 9 ) . BLI are shown ( f ( left panel ) ) . BLI signal of each mice ( f ( middle panel ) ) number of bone metastasis ( f ( right panel ) ) of every

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

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

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


Experimental procedure in vivo : MDA - MB - 231 were intracardially injected into nude mice . mice from each group at week 6 injected with control ( n = 9 ) or MDA - MB - 231 stably ovexpressed with USP8 - WT ( n = 7 ) or USP8 - CS ( n = 9 ) . Fold change of circulating EV - TβRII by ELISA 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 ( 5 × 105 per mouse ) expressing control vector or doxycycline - inducible USP8 - wt / cs tumors were grown for 2 , followed by administration of doxycycline ( n = 6 for each group ) Normalized BLI signals ( left panel ) representative mice ( right panel ) from each group at indicated times ( j ) .

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


nude mice were tail vein - injected with 4T07 - Luc ( 5 × 105 per mouse ) expressing control vector or doxycycline - inducible USP8 - wt / cs tumors were grown for 2 , followed by administration of doxycycline ( n = 6 for each group ) Immunoblot of 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 ( 5 × 105 per mouse ) expressing control vector or doxycycline - inducible USP8 - wt / cs tumors were grown for 2 , followed by administration of doxycycline ( n = 6 for each group ) ELISA of circulating exosomal TβRII level in plasma samples of mice at 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 Golgi - associated vesicle biogenesis - trafficking - related genes are significantly enriched in USP8 - high ( n = 38 ) versus USP8 - low ( n = 39 ) expressing ; shown by pre - ranked gene - set enrichment ( 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 , shows up - regulated genes upon USP8 over - expression down - regulated genes upon USP8 depletion when compared with control MDA - MB - 231 by - seq ( left panel ) . qRT - PCR of control or MDA - MB - 231 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 of EVs from 4T1 MDA - MB - 231 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 of EVs from MDA - MB - 231 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 of EVs derived from MDA - MB - 231 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 ( 5 × 105 per mouse ) expressing control shRNA or doxycycline - inducible shRNA targeting USP8 ( shUSP8 ) tumors were grown for 3 , followed by administration of doxycycline for 3 ( n = 6 for each group ) . i , of of TβRII + crEVs in plasma samples from mice at day 24 . j , Immunoblot of TEVs ( left panel ) of of TβRII + EVs ( right panel ) in plasma samples from mice at day 42 . k , Normalized photon ﬂux at indicated times . l ,

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

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 in each group at day 42 after implantation of 4T1 ( 2 × 105 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 ) bioluminescent ( right panel ) of 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 , of CD8 + T of - draining lymph nodes ( TDLN ) at day 42 .

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


b , Left : screening procedure for components critical for secretion of EV - TβRII ; Right : nanosight of GFP positive EVs derived from MDA - MB - 231 expressing TβRII - GFP 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 of total lysates derived from MDA - MB - 231 breast cancer treated with USP8 inhibitor 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 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 , shows differentially expressed genes in control DMSO - treated or USP8 inhibitor ( 5 μM ) - treated MDA - MB - 231 for 24 h ( left panel ) . Volcano plot of transcriptome proﬁles between control USP8 inhibitor treated MDA - MB - 231 ( right panel ) . Red green dots represent genes significantly up - regulated down - regulated in control MDA - MB - 231 .

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


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

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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 up - regulated epithelial - mesenchymal - transition ( EMT ) ( upper panel ) breast cancer progenitors - related gene set ( bottom panel ) are significantly enriched in control ; shown by pre - ranked gene - set enrichment ( GSEA ) .

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


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

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


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

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


k - n , Experimental procedure in vivo MDA - MB - 231 - Luc ( 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 other day for three . Normalized BLI signals ( left panel ) BLI imaging of three representive nude mice from each group at week 6 ( right panel ) were shown ( k ) . of metastasis - free survival in each experimental group followed in time ( l ) . Number of bone metastasis nodules ( m ) . of TβRII + crEVs in plasma samples from mice ( left panel ) immunoblot of metastatic nodule in mice circulating EVs from plasma ( right panel ) ( n ) .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( G ) Immunofluorescence of for TSC2 LAMP2 in presence or absence of lactate in HCT116 after serum deprivation overnight .

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


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

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


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

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


( C ) Immunofluorescence of for TSC2 LAMP2 in HCT116 degree of colocalization of TSC2 LAMP2 is graphed as mean ± . * * * p < 0 . 001 .

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


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

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


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

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


( F ) Immunofluorescence of for TSC2 LAMP2 in HCT116 degree of colocalization of TSC2 LAMP2 is graphed as mean ± . * * * p < 0 . 001 .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


E . CGH of genome stability of trisomic ES .

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


F . CGH of genome integrity of trisomic ES subclones .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


TIFA - KO were transfected with His ‑ tagged recombinant TIFA 24 h prior to H . pylori infection . Co ‑ IP with an anti ‑ His antibody was performed . Eluates total lysates were analyzed by immunoblotting using indicated antibodies . Asterisk denotes an unspecific band arrow indicates TRAF2 band .

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TIFA-KO cells were transfected with His‑tagged recombinant TIFA protein 24 h prior to H. pylori infection. Co‑IP with an anti‑His antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies. Asterisk denotes an unspecific band and the arrow indicates the TRAF2 band.


AGS ALPK1 - KO were infected with H . pylori for times shown . Co ‑ IP with an anti ‑ TRAF6 antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates total lysates were analyzed by immunoblotting using indicated antibodies .

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AGS and ALPK1-KO cells were infected with H. pylori for the times shown. Co‑IP with an anti‑TRAF6 antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS TIFA - KO were infected with H . pylori for times shown . Co ‑ IP with an anti ‑ TRAF6 antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates total lysates were analyzed by immunoblotting using indicated antibodies .

AGS and TIFA-KO cells were infected with H. pylori for the times shown. Co‑IP with an anti‑TRAF6 antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS TIFA - KO were treated with 30 ng / ml IL - 1β for times shown . Co ‑ IP with an anti ‑ TRAF6 antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates total lysates were analyzed by immunoblotting using indicated antibodies .

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AGS and TIFA-KO cells were treated with 30 ng/ml IL-1β for the times shown. Co‑IP with an anti‑TRAF6 antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


TIFA - KO were transfected with His ‑ tagged recombinant TIFA 24 h prior to H . pylori infection or treatment with 30 ng / ml LTα1β2 . Co ‑ IP with an anti ‑ His antibody was performed . Eluates total lysates were analyzed by immunoblotting using indicated antibodies .

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TIFA-KO cells were transfected with His‑tagged recombinant TIFA protein 24 h prior to H. pylori infection or treatment with 30 ng/ml LTα1β2. Co‑IP with an anti‑His antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS were pre ‑ incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by addition of ADP - heptose ( 200 nM ) . Co ‑ IP with an anti ‑ TIFA antibody was performed . Eluates total lysates were analyzed by immunoblotting using indicated antibodies . Asterisk indicates an unspecific band arrow indicates TRAF3 band .

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AGS cells were pre‑incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by addition of ADP-heptose (200 nM). Co‑IP with an anti‑TIFA antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies. Asterisk indicates an unspecific band and the arrow indicates the TRAF3 band.


AGS ALPK1 - KO were infected with H . pylori for times shown . Co ‑ IP with an anti ‑ TIFA antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates total lysates were analyzed by immunoblotting using indicated antibodies .

AGS and ALPK1-KO cells were infected with H. pylori for the times shown. Co‑IP with an anti‑TIFA antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS TIFA - KO were infected with H . pylori for times shown . Co ‑ IP with an anti ‑ TRAF2 antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates total lysates were analyzed by immunoblotting using indicated antibodies .

AGS and TIFA-KO cells were infected with H. pylori for the times shown. Co‑IP with an anti‑TRAF2 antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS were transfected with siRNA against cIAP1 . Co ‑ IP with an anti ‑ TIFA antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates total lysates were analyzed by immunoblotting using indicated antibodies .

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AGS cells were transfected with siRNA against cIAP1. Co‑IP with an anti‑TIFA antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


TIFA - KO were transfected with empty vector plasmid ( pCMV6 ) , wild ‑ type human ( phTIFA ) , phosphorylation ‑ defective mutant ( pT9A ) or TRAF6 binding ‑ defective mutant ( pE178A ) FLAG ‑ tagged TIFA plasmids . Total lysates were analyzed by immunoblotting using indicated antibodies .

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TIFA-KO cells were transfected with empty vector plasmid (pCMV6), wild‑type human (phTIFA), phosphorylation‑defective mutant (pT9A) or TRAF6 binding‑defective mutant (pE178A) FLAG‑tagged TIFA plasmids. Total cell lysates were analyzed by immunoblotting using the indicated antibodies.


TIFA - KO were transfected with empty vector plasmid ( pCMV6 ) , wild ‑ type human ( phTIFA ) , phosphorylation ‑ defective mutant ( pT9A ) or TRAF6 binding ‑ defective mutant ( pE178A ) FLAG ‑ tagged TIFA plasmids . Total lysates were separated by native gel electrophoresis under non - reducing conditions .

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TIFA-KO cells were transfected with empty vector plasmid (pCMV6), wild‑type human (phTIFA), phosphorylation‑defective mutant (pT9A) or TRAF6 binding‑defective mutant (pE178A) FLAG‑tagged TIFA plasmids. Total cell lysates were separated by native gel electrophoresis under non-reducing conditions.


In TIFA ‑ KO transiently expressing TIFA ‑ tdTomato , co - localization of TRAF6 ( A ) , TRAF2 ( B ) , cIAP1 ( C ) TRAF3 ( D ) with TIFA - tdTomato , which formed TIFAsomes upon infection with H . pylori P1 wild ‑ type , were detected by immunofluorescence . nuclei were counterstained with DAPI . Scale bar = 10 µm . TIFA ‑ KO transiently expressing TIFA ‑ tdTomato were infected with H . pylori P1 mutated in gmhA gene ( ∆ HP0857 ) . Scale bar = 10 µm .

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In TIFA‑KO cells transiently expressing TIFA‑tdTomato, co-localization of TRAF6 (A), TRAF2 (B), cIAP1 (C) and TRAF3 (D) with TIFA-tdTomato, which formed TIFAsomes upon infection with H. pylori P1 wild‑type strain, were detected by immunofluorescence staining. The nuclei were counterstained with DAPI. Scale bar = 10 µm. TIFA‑KO cells transiently expressing TIFA‑tdTomato were infected with H. pylori P1 strain mutated in the gmhA gene (∆HP0857). Scale bar = 10 µm.


( c - f ) indicated marker genes were used to select clusters for subsetting into stromal ( c ) , epithelial ( d ) , endothelial ( e ) , leukocytes ( f ) .

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(c-f) The indicated marker genes were used to select clusters for subsetting into stromal cells (c), epithelial cells (d), endothelial cells (e), and leukocytes (f).


( g - j ) heatmaps show relative gene expression for indicated marker genes for indicated stromal ( g ) , epithelial ( h ) , endothelial ( i ) , leukocyte ( j ) types .

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(g-j) The heatmaps show the relative gene expression levels for the indicated marker genes for the indicated stromal (g), epithelial (h), endothelial (i), and leukocyte (j) cell types.


( d ) Differential gene expression between end stage lung disease controls across cohorts was compared for indicated type identities . color code demonstrates similarities of gene expression changes calculated by Pearson correlation of t - value coefficient , which represents differences in likelihood of detection for any gene between health disease .

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(d) Differential gene expression between end stage lung disease patients and controls across cohorts was compared for the indicated cell type identities. The color code demonstrates similarities of gene expression changes calculated by Pearson correlation of the t-value coefficient, which represents differences in the likelihood of detection for any gene between health and disease.


( e ) shows top 79 genes differentially expressed in indicated type identities .

(e) The heatmap shows the top 79 genes differentially expressed in the indicated cell type identities.


( f - h ) box plots illustrate differences in mRNA detection for indicated genes between tissues from end stage lung fibrosis in ( f ) alveolar epithelial , ( g ) fibroblasts , ( h ) macrophages when compared to control tissue . boxes represent interquartile range , horizontal in box is median , whiskers represent 1 . 5 times interquartile range ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) .

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(f-h) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in (f) alveolar epithelial cells, (g) fibroblasts, and (h) macrophages when compared to control tissue. The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).


( e ) shows changes of indicated type signatures in published bulk - seq data ( GEO GSE124685 ) across different histopathological stages that represent increasing extent of fibrosis from stage 1 - 3 , as determined by quantitative micro - CT imaging tissue histology Samples used in this study were 10 IPF 6 control .

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(e) The heatmap shows changes of the indicated cell type signatures in published bulk RNA-seq data (GEO GSE124685) across different histopathological stages that represent increasing extent of fibrosis from stage 1-3, as determined by quantitative micro-CT imaging and tissue histology Samples used in this study were 10 IPF and 6 control patients.


( f ) heatmaps show z - scores for marker genes of indicated types across IPF stages controls .

(f) The heatmaps show z-scores for the individual marker genes of the indicated cell types across IPF stages and controls.


( b ) boxplots show number of quantified ( y - axis ) across various diagnoses ( x - axis ) . mean 10 - 90 percentiles are shown ; dotted marks 1000 ( IPF n = 16 , HP / EAA n = 8 , COP n = 11 , NSIP n = 10 , RB - IL , n = 3 , Sarcoidosis n = 22 , other ILDs n = 25 , non - IL , n = 29 ) .

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(b) The boxplots show the number of proteins quantified (y-axis) across various diagnoses (x-axis). The mean and 10-90 percentiles are shown; the dotted line marks 1000 proteins (IPF n=16, HP/EAA n=8, COP n=11, NSIP n=10, RB-IL, n=3, Sarcoidosis n=22, other ILDs n=25, non-IL, n=29).


( c Empirical cumulative density plots depict distribution of correlation coefficients for ( c ) Myofibroblast markers ( red points ) with DLCO in comparison to background ( black ) .

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(c Empirical cumulative density plots depict the distribution of correlation coefficients for (c) Myofibroblast markers (red points) with DLCO in comparison to all background proteins (black line).


d ) Empirical cumulative density plots depict distribution of correlation coefficients for ( d ) Plasma markers ( red points ) with % alveolar macrophages in BAL in comparison to background ( black ) .

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d) Empirical cumulative density plots depict the distribution of correlation coefficients for (d) Plasma cell markers (red points) with % alveolar macrophages in BAL in comparison to all background proteins (black line).


( e ) Barplot displays coefficients derived from correlating lung function associations at level with type specific ILD associations at level .

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(e) Barplot displays coefficients derived from correlating lung function associations at the protein level with cell type specific ILD associations at the RNA level.


( f ) illustrates gene expression changes associated with lung fibrosis across indicated types ( columns ) for selected BALF biomarkers ( rows ) . dotplot visualizes frequency changes of indicated types inferred from deconvolution of bulk mRNA data of ILD samples compared to controls ( GSE47460 ) . Samples used in this study from LTRC n = 254 ILD n = 108 controls .

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(f) The heatmap illustrates gene expression changes associated with lung fibrosis across indicated cell types (columns) for selected BALF protein biomarkers (rows). The dotplot visualizes the frequency changes of the indicated cell types inferred from the deconvolution of bulk mRNA data of ILD samples compared to controls (GSE47460). Samples used in this study from the LTRC n = 254 ILD patients and n = 108 controls.


( a , b ) Scatter plots stratify genes on lung function associations ( y - axis ) type specific ILD associations ( x - axis ) . of dots represents detection level of each gene in corresponding type . Colors highlight genes with marginal associations at .

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(a, b) Scatter plots stratify genes based on the protein lung function associations (y-axis) and cell type specific ILD associations (x-axis). The size of the dots represents the detection level of each gene in the corresponding cell type. Colors highlight genes with marginal associations at the protein and RNA levels.


( c , d ) box plots illustrate differences in mRNA detection for indicated genes between tissues from end stage lung fibrosis in ( c ) basal , ( d ) alveolar epithelial when compared to controls . boxes represent interquartile range , horizontal in box is median , whiskers represent 1 . 5 times interquartile range ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) .

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(c, d) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in (c) basal cells, and (d) alveolar epithelial cells when compared to controls. The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).


( e , f ) scatter plots show positive correlation of indicated in BALF ( MS - , x - axis ) with meta lung function ( y - axis ) .

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(e, f) The scatter plots show the positive correlation of the indicated proteins in BALF (MS-intensity, x-axis) with meta lung function (y-axis).


( g - j ) Immunofluorescence of indicated type markers in IPF ( n = 3 ) control samples ( n = 3 ) .

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(g-j) Immunofluorescence analysis of the indicated proteins and cell type markers in IPF (n=3) and control samples (n=3).


( a ) scatter plots show positive correlation of CFHR1 in BALF ( MS - , x - axis ) with meta lung function ( y - axis ) .

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(a) The scatter plots show the positive correlation of CFHR1 in BALF (MS-intensity, x-axis) with meta lung function (y-axis).


( c ) volcano plot depicts fold changes ( x - axis ) - log10 p - values ( y - axis ) derived from differential gene expression using diffxpy between ILD controls within subset of PDGFRB + pericytes ( outlier values truncated ) .

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(c) The volcano plot depicts the fold changes (x-axis) and the -log10 p-values (y-axis) derived from the differential gene expression analysis using diffxpy between ILD and controls within the subset of PDGFRB+ pericytes (outlier values truncated).


( e ) shows expression of genes most strongly associated with SSTR2 expression within pericytes ( left color bar indicates significant correlation or anti - correlation ) .

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(e) The heatmap shows the expression of genes most strongly associated with SSTR2 expression within the pericytes (left color bar indicates significant correlation or anti-correlation).


( f ) Immunofluorescence of SSTR2 , pericyte type marker PDGFRB a marker for smooth muscle DESMIN in IPF ( n = 3 ) control samples ( n = 3 ) .

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(f) Immunofluorescence analysis of SSTR2, the pericyte cell type marker PDGFRB and a marker for smooth muscle cells DESMIN in IPF (n=3) and control samples (n=3).


( g ) Representative of immunohistochemistry of SSTR2 expression in tissue regions ( n = 474 ) from ILD ( n = 53 ) control ( n = 26 ) .

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(g) Representative images of immunohistochemistry analysis of SSTR2 protein expression in tissue regions (n = 474) from ILD patients (n = 53) and control patients (n = 26).


( h ) Correlation of immunohistochemistry signal of SSTR2 with Ashcroft scores .

(h) Correlation of immunohistochemistry signal of SSTR2 with Ashcroft scores.


( i ) bar graph shows genes most strongly correlated with SSTR2 belonging to GO category " transcription regulators " . dotted marks a correlation coefficient of zero .

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(i) The bar graph shows the genes most strongly correlated with SSTR2 belonging to the GO category "transcription regulators". The dotted line marks a correlation coefficient of zero.


( c ) Relative expression level of CRTAC1 across human organs .

(c) Relative expression level of CRTAC1 across human organs.


( d ) box plots illustrate differences in mRNA detection for CRTAC1 in alveolar epithelial from fibrosis compared to control samples across three indicated cohorts ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) .

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(d) The box plots illustrate differences in mRNA detection for CRTAC1 in alveolar epithelial cells from fibrosis patients compared to control samples across the three indicated patient cohorts (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).


( e ) Relative gene expression of CRTAC1 in GSE47460 . Dots represent average expression in tissue of . represents mean , error bars show SD . CRTAC1 is significantly downregulated in ILD but not COPD

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(e) Relative gene expression levels of CRTAC1 in GSE47460. Dots represent average expression in the tissue of individual patients. The line represents the mean, error bars show SD. CRTAC1 is significantly downregulated in ILD but not COPD patients


( f ) For each single cohort , gene - gene correlations with CRTAC1 within SFTPC + AT - 2 were calculated . indicated genes were selected on their common direction of correlation across cohorts .

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(f) For each single cell cohort, the gene-gene correlations with CRTAC1 within the SFTPC+ AT-2 cells were calculated. The indicated genes were selected based on their common direction of correlation across cohorts.


( h ) bar graph shows gene categories most strongly correlated with CRTAC1 belonging to GO category of " transcription regulators " . dotted marks a correlation coefficient of zero .

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(h) The bar graph shows the gene categories most strongly correlated with CRTAC1 belonging to the GO category of "transcription regulators". The dotted line marks a correlation coefficient of zero.


( n ) Immunofluorescence of SPRR1A , KRT8 as well as SFTPC in IPF ( n = 3 ) control samples ( n = 2 ) .

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(n) Immunofluorescence analysis of SPRR1A, KRT8 as well as SFTPC in IPF (n=3) and control samples (n=2).


( o ) A high throughput experimental workflow for plasma proteomics ( Niu allowed for profiling of two independent cohorts of ILD ( Munich , n = 30 Hannover , n = 81 ; healthy matched controls , n = 30 ) . quantified in plasma are shown , ranked by their abundance measured by mass spectrometry ( MS - ) .

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(o) A high throughput experimental workflow for plasma proteomics(Niu allowed for profiling of two independent cohorts of ILD patients (Munich, n=30 and Hannover, n=81; healthy age matched controls, n=30). All proteins quantified in plasma are shown, ranked by their abundance measured by mass spectrometry (MS-intensity).


( p ) indicated from plasma were selected on their common direction of correlation with lung function in two independent cohorts with distinct clinical characteristics .

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(p) The indicated proteins from the plasma analysis were selected based on their common direction of correlation with patient lung function in two independent patient cohorts with distinct clinical characteristics.


( q ) shows predicted relative contribution of lung types to association of biomarker signatures in plasma with lung function ( forced vital capacity - FVC ) . were split in two groups , one with a mild decline in lung function [ FVC 60 - 100 % ] one with severe loss of lung function [ FVC 20 - 60 % ] compared to healthy matched controls .

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(q) The heatmap shows the predicted relative contribution of lung cell types to the association of protein biomarker signatures in plasma with lung function (forced vital capacity - FVC). Patients were split in two groups, one with a mild decline in lung function [FVC 60-100%] and one with severe loss of lung function [FVC 20-60%] and compared to healthy age matched controls.


( B ) for CDC50A RIM1 on cultured neurons at DIV21 . left panel shows CDC50A is expressed in both neuronal soma processes . right panel exhibits CDC50A colocalize to pre - synapses alongside neuronal processes . MAP2 , Microtubule - associated 2 , was used to identify neuronal trace dendritic processes . RIM1 is an active zone , which marks pre - synapses . Left scale bar = 50 µm ; right scale bar = 2 µm . ( C ) of of RIM1 + pre - synapses colocalizing to CDC50A . N = 3 for biological replicates .

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(B) IHC for CDC50A and RIM1 on cultured neurons at DIV21. The left panel shows CDC50A is expressed in both neuronal soma and processes. The right panel exhibits CDC50A colocalize to pre-synapses alongside neuronal processes. MAP2, Microtubule-associated protein 2, was used to identify neuronal cells and trace dendritic processes. RIM1 is an active zone protein, which marks pre-synapses. Left scale bar = 50 µm; right scale bar = 2 µm. (C) Quantification of the percentage of RIM1+ pre-synapses colocalizing to CDC50A. N = 3 for biological replicates.


( E ) for CDC50A vGluT2 in developmental dLGN at P14 , P21 P30 . left panels show overview of immunostaining in dLGN , scale bar = 200 µm . right panels are zoomed - in , scale bar = 5 µm . Arrows indicate colocalized CDC50A vGluT2 . ( F ) of of CDC50A + / vGlut2 + synapses . result shows that a significantly higher of CDC50A + / vGlut2 + synapses at P21 P30 than P14 . N = 6 for biological replicates .

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(E) IHC for CDC50A and vGluT2 in the developmental dLGN at P14, P21 and P30. The left panels show the overview of immunostaining in the dLGN, scale bar = 200 µm. The right panels are the zoomed-in images, scale bar = 5 µm. Arrows indicate the colocalized CDC50A and vGluT2. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. The result shows that a significantly higher percentage of CDC50A+/vGlut2+ synapses at P21 and P30 than P14. N = 6 for biological replicates.


( C ) for CDC50A . CDC50A intensities were reduced after neuronal inactivation ( day 7 ) , was recovered after removing neuronal inhibitors ( day 13 ) . Scale bar , 10 µm . ( D ) Relative CDC50A intensities of neurons . N = 5 for biological replicates , mean ± SD , * p < 0 . 5 , * * * * p < 0 . 0001 by one - way ANOVA with Holm - Sidak ' s multiple comparisons .

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(C) IHC for CDC50A. CDC50A protein intensities were reduced after neuronal inactivation (day 7), and was recovered after removing neuronal inhibitors (day 13). Scale bar, 10 µm. (D) Relative CDC50A intensities of neurons. N = 5 for biological replicates, mean ± SD, * p < 0.5, **** p < 0.0001 by one-way ANOVA with Holm-Sidak's multiple comparisons test.


( E ) CDC50A expression at RGC inputs after 5 - day consecutive TTX treatment . Scale bar , 10 µm . ( F ) of of CDC50A + / vGlut2 + synapses . N = 5 for biological replicates , mean ± SD , * * * * p < 0 . 0001 by Student ' s .

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(E) CDC50A expression at RGC inputs after 5-day consecutive TTX treatment. Scale bar, 10 µm. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. N = 5 for biological replicates, mean ± SD, **** p < 0.0001 by Student's test.


( A ) Immunostaining of CDC50A on Cdc50a knockdown neurons controls . Accell Cdc50a siRNA pool was used to knock down Cdc50a in neurons for 7 days . CDC50A expression alongside neuronal processed was reduced after Cdc50a siRNA treatment . Scale bar , 10 µm .

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(A) Immunostaining of CDC50A on Cdc50a knockdown neurons and controls. Accell Cdc50a siRNA pool was used to knock down Cdc50a in neurons for 7 days. CDC50A expression alongside neuronal processed was reduced after Cdc50a siRNA treatment. Scale bar, 10 µm.


( D ) of Cdc50a - RNAi neurons exhibited pSIVA colocalized with synapsin I , a general synapse marker . dotted square was zoomed - in shown on right side . Arrows indicated where pSIVA colocalized with synapsin I . Scale bar , 5 µm .

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(D) IHC of Cdc50a-RNAi neurons exhibited pSIVA colocalized with synapsin I, a general synapse marker. The dotted square was zoomed-in shown on the right side. Arrows indicated where pSIVA colocalized with synapsin I. Scale bar, 5 µm.


( D ) Representative of CDC50A expression at synapses . Scale bar , 5 µm . Arrows indicate VGluT2 + RGC inputs . ( E ) Relative CDC50A expression at mCherry + synapses in Cdc50a RNAi mice controls . N = 3 for biological replicates , mean ± SD , * p < 0 . 05 by Student ' s t - .

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(D) Representative IHC images of CDC50A expression at synapses. Scale bar, 5 µm. Arrows indicate VGluT2+ RGC inputs. (E) Relative CDC50A expression at mCherry+ synapses in Cdc50a RNAi mice and controls. N = 3 for biological replicates, mean ± SD, * p < 0.05 by Student's t-test.


( F ) PSIVA in dLGN . Exposed PS was detected by pSIVA on RGC inputs in Cdc50a - RNAi mice . Scale bar , 5 µm . Arrows indicate pSIVA + / mCherry + RGC inputs . ( G ) of of pSIVA + / mCherry + RGC inputs in total mCherry + RGC inputs . N = 4 for biological replicates , mean ± , * * p < 0 . 01 by Student ' s t - .

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(F) PSIVA staining in the dLGN. Exposed PS was detected by pSIVA on RGC inputs in Cdc50a-RNAi mice. Scale bar, 5 µm. Arrows indicate pSIVA+/mCherry+ RGC inputs. (G) Quantification of the percentage of pSIVA+/mCherry+ RGC inputs in total mCherry+ RGC inputs. N = 4 for biological replicates, mean ± SEM, **p < 0.01 by Student's t-test.


( F ) Maximum z - projection of vGluT2 + synapses in region of Cdc50a RNAi mice controls . Right panel showed enlarged synapses in boxed region . Scale bar , 20 µm .

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(F) Maximum intensity z-projection images of vGluT2+ synapses in the core region of Cdc50a RNAi mice and controls. Right panel showed enlarged synapses in the boxed region. Scale bar, 20 µm.