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 .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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


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 .

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 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 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 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 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 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 .

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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 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 .

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

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 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 .

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

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 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 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 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 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 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 .

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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 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 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 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 .

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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 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 .

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

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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 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 .

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

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


( 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 ) .

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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 ) 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 .

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

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


( 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 ) .

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

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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 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 ) .

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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 the protein level with cell type specific ILD associations at the RNA 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 ) 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 .

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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 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 .

(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 ) 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 ) .

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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 ) The scatter plots show the positive correlation of the indicated proteins in BALF ( MS - intensity , 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 analysis of the indicated proteins and cell type markers in IPF ( n = 3 ) and 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 ) The scatter plots show the positive correlation of CFHR1 in BALF ( MS - intensity , 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 ) 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 ) .

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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 ) 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 ) .

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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 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 ) .

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

(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 ) 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 .

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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 ) 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 ) .

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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 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

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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 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 .

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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 ) 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 .

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

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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 ) 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 .

(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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) Quantification of the percentage of CDC50A + / vGlut2 + synapses . N = 5 for biological replicates , mean ± SD , * * * * p < 0 . 0001 by Student ' s test .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


( H ) Maximum intensity z - projection images of vGluT2 + synapses in the shell region . Enlarged images ( right panel ) shows large " complex encapsulated " boutons . Scale bar , 20 µm . ( I ) Quantification of vGluT2 + synapse density . N = 3 for biological replicates , mean ± SD , Student ' s t - test .

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(H) Maximum intensity z-projection images of vGluT2+ synapses in the shell region. Enlarged images (right panel) shows large "complex encapsulated" boutons. Scale bar, 20 µm. (I) Quantification of vGluT2+ synapse density. N = 3 for biological replicates, mean ± SD, Student's t-test.


( D ) Images of CTB488 and CTB647 labeling of RGC inputs in the dLGN . Scale bar , 20 µm . ( E ) Quantification of CTB488 + inputs ( non - infected areas ) versus CTB647 + inputs ( infected areas ) in Cdc50a RNAi mice and controls . N = 3 for biological replicates , mean ± SEM , * * * * p < 0 . 0001 , two - way ANOVA with Sidak ' s multiple comparisons test .

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(D) Images of CTB488 and CTB647 labeling of RGC inputs in the dLGN. Scale bar, 20 µm. (E) Quantification of CTB488+ inputs (non-infected areas) versus CTB647+ inputs (infected areas) in Cdc50a RNAi mice and controls. N = 3 for biological replicates, mean ± SEM, **** p < 0.0001, two-way ANOVA with Sidak's multiple comparisons test.


( A ) The confocal images , 3D - rendered images and orthogonal views of microglia engulfing vGluT2 + synapses in controls and Cdc50a - RNAi mice . Scale bar , 10 µm .

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(A) The confocal images, 3D-rendered images and orthogonal views of microglia engulfing vGluT2+ synapses in controls and Cdc50a-RNAi mice. Scale bar, 10 µm.


( A ) IHC images for vGluT2 staining in the dLGN of microglial Gpr56 conditional knockout mice ( Gpr56fl / fl ; Cx3cr1cre / + ) and controls ( Gpr56 + / + ; Cx3cr1cre / + ) . Both mice were intravitreally injected with AAV2 carrying Cdc50a shRNA . The yellow squares indicated the AAV2 non - infected areas and the dotted outlines indicated the AAV2 infected areas . Scale bar , 200 µm .

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(A) IHC images for vGluT2 staining in the dLGN of microglial Gpr56 conditional knockout mice (Gpr56fl/fl; Cx3cr1cre/+) and controls (Gpr56+/+; Cx3cr1cre/+). Both mice were intravitreally injected with AAV2 carrying Cdc50a shRNA. The yellow squares indicated the AAV2 non-infected areas and the dotted outlines indicated the AAV2 infected areas. Scale bar, 200 µm.


( B ) Confocal images of vGluT2 + synapses in the AAV2 non - infected areas and infected areas of the dLGN . Scale bar , 20 µm . ( C ) Quantification of vGluT2 + synapses in the non - infected areas and infected areas of microglial GPR56 conditional knockout mice and controls . N = 4 for biological replicates , mean ± SD , * p < 0 . 05 , * * * * p < 0 . 0001 by two - way ANOVA with Sidak ' s multiple comparisons test .

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(B) Confocal images of vGluT2+ synapses in the AAV2 non-infected areas and infected areas of the dLGN. Scale bar, 20 µm. (C) Quantification of vGluT2+ synapses in the non-infected areas and infected areas of microglial GPR56 conditional knockout mice and controls. N = 4 for biological replicates, mean ± SD, * p<0.05, **** p < 0.0001 by two-way ANOVA with Sidak's multiple comparisons test.


Verification of PTPδ - tdTomato reporter mice by Western blot analysis of whole - brain lysates from heterozygote ( HT ) and homozygous ( Ho ) mice ( P21 ) and PTPδ using mCherry antibodies . Note that levels of endogenous PTPδ protein ( ~ 85 kDa ) are decreased , and that PTPδ - tdTomato protein ( ~ 140 kDa ) is detectable only in reporter mice ( HT and Ho ) . Note also that the 85 - kDa band represents the transmembrane domain + cytoplasmic fragment of the full - length PTPδ protein that undergoes proteolytic cleavage at extracellular membrane - proximal sites , which can only be recognized by the PTPδ C - terminal antibody that targets the cytoplasmic region . The PTPδ - tdTomato protein ( ~ 140 kDa ) contains the membrane domain + cytoplasmic fragment of PTPδ ( ~ 85 kDa ) fused to tdTomato ( 55 kDa ) . The N - terminal antibody recognizes equal amounts of the C - terminal cleavage product derived from PTPδ and PTPδ - tdTomato proteins .

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Verification of PTPδ-tdTomato reporter mice by Western blot analysis of whole-brain lysates from heterozygote (HT) and homozygous (Ho) mice (P21) and PTPδ using mCherry antibodies. Note that levels of endogenous PTPδ protein (~85 kDa) are decreased, and that PTPδ-tdTomato protein (~140 kDa) is detectable only in reporter mice (HT and Ho). Note also that the 85-kDa band represents the transmembrane domain + cytoplasmic fragment of the full-length PTPδ protein that undergoes proteolytic cleavage at extracellular membrane-proximal sites, which can only be recognized by the PTPδ C-terminal antibody that targets the cytoplasmic region. The PTPδ-tdTomato protein (~140 kDa) contains the membrane domain + cytoplasmic fragment of PTPδ (~85 kDa) fused to tdTomato (55 kDa). The N-terminal antibody recognizes equal amounts of the C-terminal cleavage product derived from PTPδ and PTPδ-tdTomato proteins.