( G ) Ambra1 knockdown declines Beclin 1 ubiquitination . HeLa cells with Ambra1 knockdown were treated with EBSS for 1 h , followed by immunoprecipitated with anti ‐ Beclin 1 antibody . Immunoprecipitates were dissociated with 1 % SDS and re ‐ immunoprecipitated with anti ‐ Beclin 1 antibody , followed by immunoblotting with the indicated antibodies .

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(G) Ambra1 knockdown declines Beclin 1 ubiquitination. HeLa cells with Ambra1 knockdown were treated with EBSS for 1 h, followed by immunoprecipitated with anti‐Beclin 1 antibody. Immunoprecipitates were dissociated with 1% SDS and re‐immunoprecipitated with anti‐Beclin 1 antibody, followed by immunoblotting with the indicated antibodies.


( H ) Ambra1 overexpression increases Beclin 1 ubiquitination . HeLa cells with Ambra1 overexpression were treated as above .

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(H) Ambra1 overexpression increases Beclin 1 ubiquitination. HeLa cells with Ambra1 overexpression were treated as above.


( I ) Ambra1 can directly ubiquitinate Beclin 1 by an in vitro ubiquitination assay . Recombinant Beclin 1 ( rBeclin 1 ) and components of the ubiquitination system were expressed and purified as described in Materials and methods . The reaction started by mixing the indicated components in ubiquitination buffer at 37 ° C for 1 h and followed by immunoblotting .

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(I) Ambra1 can directly ubiquitinate Beclin 1 by an in vitro ubiquitination assay. Recombinant Beclin 1 (rBeclin 1) and components of the ubiquitination system were expressed and purified as described in Materials and methods. The reaction started by mixing the indicated components in ubiquitination buffer at 37°C for 1 h and followed by immunoblotting.


( J , K ) WASH and Ambra1 competitively bind to Beclin 1 . HeLa cells with vector or WASH overexpression were starved for 1 h and lysed for immunoprecipitation with anti ‐ Beclin 1 antibody ( J ) . Recombinant Ambra1 and WASH proteins were incubated with GST - Beclin 1 , followed by GST pull ‐ down assays ( K ) . Scale bar , 10 μm . Data are shown as means ± s . d . * * P0 . 01 and * * * P0 . 001 . Data were repeated for at least three times with similar results . Source data for this figure is available on the online supplementary information page .

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(J, K) WASH and Ambra1 competitively bind to Beclin 1. HeLa cells with vector or WASH overexpression were starved for 1 h and lysed for immunoprecipitation with anti‐Beclin 1 antibody (J). Recombinant Ambra1 and WASH proteins were incubated with GST-Beclin 1, followed by GST pull‐down assays (K). Scale bar, 10 μm. Data are shown as means±s.d. **P0.01 and ***P0.001. Data were repeated for at least three times with similar results.Source data for this figure is available on the online supplementary information page.


( C ) NSI mice homozygous for the Fah knockout displayed a lethal neonatal phenotype ( left ) . Fah expression was detected in the livers of homozygous NSI mice with Fah knockout and Fah intact NSI mice by Western blotting ( right ) . Cropped blots are

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(C) NSI mice homozygous for the Fah knockout displayed a lethal neonatal phenotype (left). Fah expression was detected in the livers of homozygous NSI mice with Fah knockout and Fah intact NSI mice by Western blotting (right). Cropped blots are shown


( F ) Representative PCR amplification of MYC , TP53R249S , KRASG12D , NRASG12D , CTNNB1S45F , BRAFV600E , AXIN1G652S , IL6 , PIK3CAE542K and CSF1RY969C in the genomic DNA of dissected primary tumors harvested from NSIF mice transplanted with OC - PHHs ( Tumor ) and their corresponding overexpression vector plasmids as positive controls ( OCs )

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(F) Representative PCR amplification of MYC, TP53R249S, KRASG12D, NRASG12D, CTNNB1S45F, BRAFV600E, AXIN1G652S, IL6, PIK3CAE542K and CSF1RY969C in the genomic DNA of dissected primary tumors harvested from NSIF mice transplanted with OC-PHHs (Tumor) and their corresponding overexpression vector plasmids as positive controls (OCs)


( I ) Expression of MYC , p53 , and KRASG12D was detected in the iHCC samples ( n = 3 , biological replicates ) by Western blotting . Cropped blots are

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(I) Expression of MYC, p53, and KRASG12D was detected in the iHCC samples (n=3, biological replicates) by Western blotting. Cropped blots are shown


( B ) Genomic DNA of colonies that were derived from single cells of iHCC1 - 1 and iHCC2 - 1 tumor samples were extracted for PCR amplification of MYC , TP53R249S , and

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(B) Genomic DNA of colonies that were derived from single cells of iHCC1-1 and iHCC2-1 tumor samples were extracted for PCR amplification of MYC, TP53R249S, and KRASG12D


( B ) Volcano plot of RNA - seq data showing genes with differential expression ( 1 , 591 upregulated and 2 , 243 downregulated genes , fold change ≥ 2 and P value ≤ 0 . 05 ) in iHCC cells compared to PHHs . TP53 , KRAS , MYC , MUC1 , and AFP were highly expressed in iHCC cells , whereas hepatocyte signature markers , including FOXA2 , FOXA3 , ATF5 , HNF4A , and ALB were highly expressed in

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(B) Volcano plot of RNA-seq data showing genes with differential expression (1,591 upregulated and 2,243 downregulated genes, fold change≥2 and P value≤0.05) in iHCC cells compared to PHHs. TP53, KRAS, MYC, MUC1, and AFP were highly expressed in iHCC cells, whereas hepatocyte signature markers, including FOXA2, FOXA3, ATF5, HNF4A, and ALB were highly expressed in PHHs


( D ) Heat map analysis of the log2 ( RPKM ) values of selected genes found to be highly expressed or downregulated in PHHs and iHCC cells . These genes were classified into three categories based on their

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(D) Heat map analysis of the log2(RPKM) values of selected genes found to be highly expressed or downregulated in PHHs and iHCC cells. These genes were classified into three categories based on their functions


( A ) Heat map shows the top 20 genes encoding cell surface markers found to be highly expressed in iHCC samples compared to the corresponding PHHs . Within these 20 genes , seven genes highlighted in red were selected because their expression was correlated with poor prognosis of HCC patients based on TCGA - HCC ( TCGA - LIHC )

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(A) Heat map shows the top 20 genes encoding cell surface markers found to be highly expressed in iHCC samples compared to the corresponding PHHs. Within these 20 genes, seven genes highlighted in red were selected because their expression was correlated with poor prognosis of HCC patients based on TCGA-HCC (TCGA-LIHC) analysis


( B ) Representative cytotoxicity CARFAP - T and CARMUC1 - T cells assessed by coincubation with iHCC2 - 1 cells . P = 0 . 0038 ( CARFAP - T vs . mock T ) , and P < 0 . 0478 ( CARMUC1 - T vs . mock - T ) . P values were determined by paired t - test , * * P ≤ 0 . 05 . Cells were seeded at E : T ratios ranging from 4 : 1 to 1 : 4 ; data are presented as the mean ± SD . N - numbers refer to biological replicates ( n = 3 )

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(B) Representative cytotoxicity CARFAP-T and CARMUC1-T cells assessed by coincubation with iHCC2-1 cells. P=0.0038 (CARFAP-T vs. mock T), and P<0.0478 (CARMUC1-T vs. mock-T). P values were determined by paired t-test, **P≤0.05. Cells were seeded at E: T ratios ranging from 4:1 to 1:4; data are presented as the mean ± SD. N-numbers refer to biological replicates (n=3)


( D ) Clonogenic assay of iHCC1 - 1 cells treated with a MEK inhibitor ( trametinib ) and / or a CDK4 / 6 inhibitor ( palbociclib ) ; Representative images were shown based on three biological

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(D) Clonogenic assay of iHCC1-1 cells treated with a MEK inhibitor (trametinib) and/or a CDK4/6 inhibitor (palbociclib); Representative images were shown based on three biological replicates


( F ) Clonogenic assays ( Top ) of iHCC1 - 1 cells treated with XL413 . The data are the representative of three biological replications . ( Bottom ) Growth curves of iHCC in xenografts ( n = 5 ) treated with PBS or XL413 at 50 mm3 tumor volume . P = 0 . 0486 ( XL413 vs . PBS group ) by two - way ANOVA with Sidak ' s multiple comparison test at Day24 , * P ≤ 0 . 05 . Data are presented as the mean ± SD . N - numbers refer to biological replicates .

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(F) Clonogenic assays (Top) of iHCC1-1 cells treated with XL413. The data are the representative of three biological replications. (Bottom) Growth curves of iHCC in xenografts (n=5) treated with PBS or XL413 at 50 mm3 tumor volume. P=0.0486 (XL413 vs. PBS group) by two-way ANOVA with Sidak's multiple comparison test at Day24, *P≤0.05. Data are presented as the mean ± SD. N-numbers refer to biological replicates.


( D ) ACE2 levels were characterized using RNAscope . A representative 3D view of a 232 x 232 μm2 field of view of the AT layer of an uninfected control at 3 days at air - liquid interface ( 3C ) is shown . The nuclear labelling is indicated in electric indigo and ACE2 mRNA is labelled in spring green LUT . ( G ) Same as in ( D ) with infected LoC at 3 dpi ( G ) shown .

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(D) ACE2 levels were characterized using RNAscope. A representative 3D view of a 232 x 232 μm2 field of view of the AT layer of an uninfected control at 3 days at air-liquid interface (3C) is shown. The nuclear labelling is indicated in electric indigo and ACE2 mRNA is labelled in spring green LUT. (G) Same as in (D) with infected LoC at 3 dpi (G) shown.


( I , J ) A plot of the fold change in expression of host proteins required for viral entry in the AT and endothelial layer of LoCs at 1 day - post infection ( n = 4 biological replicates ) and 3 days - post infection ( n = 3 biological replicates ) vs . uninfected control LoCs ( n = 3 biological replicates ) . Data information : The bars represent the mean value , the solid line represents the median value , and the error bars represent the standard deviation . P - values are calculated using a Kruskal - Wallis one - way ANOVA test , * represents p < = 0 . 05 , * * represents p < = 0 . 01 , * * represents p < = 0 . 001 . Scale bar = 20 μm .

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(I, J) A plot of the fold change in expression of host proteins required for viral entry in the AT and endothelial layer of LoCs at 1 day-post infection (n=4 biological replicates) and 3 days-post infection (n=3 biological replicates) vs. uninfected control LoCs (n=3 biological replicates). Data information: The bars represent the mean value, the solid line represents the median value, and the error bars represent the standard deviation. P-values are calculated using a Kruskal-Wallis one-way ANOVA test, * represents p<=0.05, ** represents p<=0.01, ** represents p<=0.001. Scale bar = 20 μm.


( A The kinetics of release of SARS - CoV - 2 progeny was assessed from samples obtained from a wash of the apical face of the chip and the vascular effluent ( A ) collected at each day - post infection . ( A ) The number of viral genomes in each sample was quantified using a one - step qRT - PCR assay for the SARS - CoV - 2 N gene . The starting inoculum introduced to the apical side ( labelled ' Inn ' ) corresponded to 200 - 300 PFU . Lines join datapoints from the same LoC , reconstituted with ( green squares ) and without ( dark blue circles ) macrophages . Viral genome numbers within the apical wash showed a declining trend over 3 days of infection . Viral genomes are not detected ( ' nd ' ) in the vascular effluent for all the chips shown . Data information : In all plots , the bars represent the mean value , and the error bars represent the standard deviation , the solid black line in ( A ) represents the median .

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(A The kinetics of release of SARS-CoV-2 progeny was assessed from samples obtained from a wash of the apical face of the chip and the vascular effluent (A) collected at each day-post infection. (A) The number of viral genomes in each sample was quantified using a one-step qRT-PCR assay for the SARS-CoV-2 N gene. The starting inoculum introduced to the apical side (labelled 'Inn') corresponded to 200-300 PFU. Lines join datapoints from the same LoC, reconstituted with (green squares) and without (dark blue circles) macrophages. Viral genome numbers within the apical wash showed a declining trend over 3 days of infection. Viral genomes are not detected ('nd') in the vascular effluent for all the chips shown. Data information: In all plots, the bars represent the mean value, and the error bars represent the standard deviation, the solid black line in (A) represents the median.


( A - D ) 3D views of representative 232 x 232 μm2 ( A , B ) and 155 x 155 μm2 ( C , D ) fields of view from confocal imaging of an uninfected control ( A ) , and infected chips without macrophages at days 2 ( B ) and 3 ( C , D ) post - infection , respectively . Actin and nuclear labelling are shown in azure and indigo , respectively . ( B ) At 2 dpi , adjacent areas of endothelial cell clusters identified by a stronger nuclear stain and brighter actin staining ( yellow arrows ) and cells with increased permeability and loss of confluence ( white arrows ) are indicated . ( C ) At 3 dpi , a significant loss of barrier integrity is observed in the endothelial layer , whereas the AT layer directly above this field of view ( D ) is relatively intact . A small area of missing cells in the AT layer is indicated with a white arrow in ( D ) .

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(A-D) 3D views of representative 232 x 232 μm2 (A, B) and 155 x 155 μm2 (C, D) fields of view from confocal imaging of an uninfected control (A), and infected chips without macrophages at days 2 (B) and 3 (C, D) post-infection, respectively. Actin and nuclear labelling are shown in azure and indigo, respectively. (B) At 2 dpi, adjacent areas of endothelial cell clusters identified by a stronger nuclear stain and brighter actin staining (yellow arrows) and cells with increased permeability and loss of confluence (white arrows) are indicated. (C) At 3 dpi, a significant loss of barrier integrity is observed in the endothelial layer, whereas the AT layer directly above this field of view (D) is relatively intact. A small area of missing cells in the AT layer is indicated with a white arrow in (D).


( G - J ) The fields of view in ( C , D ) are shown also in ( G , H ) respectively , SARS - CoV - 2 spike ( S ) proteins labelled via immunofluorescence are false - colored in amber . Zooms in ( I ) and ( J ) show clear evidence of intracellular spike protein within both endothelial and ATs , respectively .

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(G-J) The fields of view in (C, D) are shown also in (G, H) respectively, SARS-CoV-2 spike (S) proteins labelled via immunofluorescence are false-colored in amber. Zooms in (I) and (J) show clear evidence of intracellular spike protein within both endothelial and ATs, respectively.


( A - D ) Maximum intensity projects of 232 x 232 μm2 fields of view from the AT ( A , B ) and endothelial layers of an uninfected control LoC . ATs show strong actin ( A ) and TJP1 expression ( B , identified via antibody staining ) at the cell junction boundaries . Endothelial cells also show high levels of TJP - 1 expression ( C ) together with CD31 expression at cell junctions ( D , identified via antibody labelling ) . Actin , TJP - 1 , CD31 , and nuclear labelling are indicated with the azure , bright pink , spring green and electric indigo LUTs , respectively .

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(A-D) Maximum intensity projects of 232 x 232 μm2 fields of view from the AT (A, B) and endothelial layers of an uninfected control LoC. ATs show strong actin (A) and TJP1 expression (B, identified via antibody staining) at the cell junction boundaries. Endothelial cells also show high levels of TJP-1 expression (C) together with CD31 expression at cell junctions (D, identified via antibody labelling). Actin, TJP-1, CD31, and nuclear labelling are indicated with the azure, bright pink, spring green and electric indigo LUTs, respectively.


( E - H ) 3D views of one 232 x 232 μm2 field of view of the AT layer ( E ) and ( F - H ) three 232 x 232 μm2 fields of view of the endothelial layer of an infected LoC at 1 dpi . Areas with lower TJP1 expression in the AT layer in ( E ) are indicated by white arrows . ( F - H ) Areas with loss of tight junctions between cells on the endothelial surface is indicated by white arrows . The proportion of the surface area covered by confluent cells decreases sequentially from ( F ) to ( H ) . Endothelial cell clusters with low CD31 expression are clearly visible , and the proportion of the surface area covered by these clusters increases sequentially from ( F ) to ( H ) . In ( F - H ) , spike protein is identified via an anti - SARS - spike antibody and shown in amber LUT , and von Willebrand Factor identified by antibody labelling is shown in bring pink LUT .

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(E-H) 3D views of one 232 x 232 μm2 field of view of the AT layer (E) and (F-H) three 232 x 232 μm2 fields of view of the endothelial layer of an infected LoC at 1 dpi. Areas with lower TJP1 expression in the AT layer in (E) are indicated by white arrows. (F-H) Areas with loss of tight junctions between cells on the endothelial surface is indicated by white arrows. The proportion of the surface area covered by confluent cells decreases sequentially from (F) to (H). Endothelial cell clusters with low CD31 expression are clearly visible, and the proportion of the surface area covered by these clusters increases sequentially from (F) to (H). In (F-H), spike protein is identified via an anti-SARS-spike antibody and shown in amber LUT, and von Willebrand Factor identified by antibody labelling is shown in bring pink LUT.


( I - L ) Zooms of two endothelial cell clusters ( I , J and K , L ) in the endothelial layer of another infected LoC at 1 dpi confirm that little or no CD31 expression is detected ( I , K ) despite high levels of actin and TJP - 1 protein expression in these cells . ( J , L ) .

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(I-L) Zooms of two endothelial cell clusters (I, J and K, L) in the endothelial layer of another infected LoC at 1 dpi confirm that little or no CD31 expression is detected (I, K) despite high levels of actin and TJP-1 protein expression in these cells. (J, L).


( M ) Plot of the fold change in expression of TJP1 ( AT and endothelial layers ) and CDH5 , CD31 , and CD146 ( endothelial layers only ) in infected LoCs at 1 and 3 dpi ( n = 3 biological replicates in each case ) vs , uninfected controls ( n = 3 biological replicates ) . ( N ) Plot of the fold change in gene expression of endothelial cell specific coagulation markers - Tissue Factor ( F3 ) , Tissue Factor Pathway Inhibitor ( TFPI ) , Thrombomodulin ( THBD ) , Plasminogen Activator Inhibitor ( SERPINE1 ) and von Willebrand Factor ( VWF ) in infected LoCs at 1 and 3 dpi ( n = 3 biological replicates in each case ) vs . uninfected controls ( n = 3 biological replicates ) . Data information : In all plots , bars represent the mean value and error bars represent the standard deviation . Scale bar = 20 μm . P - values are calculated using a one - way Kruskal - Wallis ANOVA test , * represents p ≤ 0 . 05 , * * represents p ≤ 0 . 01 , * * represents p ≤ 0 . 001 .

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(M) Plot of the fold change in expression of TJP1 (AT and endothelial layers) and CDH5, CD31, and CD146 (endothelial layers only) in infected LoCs at 1 and 3 dpi (n=3 biological replicates in each case) vs, uninfected controls (n=3 biological replicates). (N) Plot of the fold change in gene expression of endothelial cell specific coagulation markers - Tissue Factor (F3), Tissue Factor Pathway Inhibitor (TFPI), Thrombomodulin (THBD), Plasminogen Activator Inhibitor (SERPINE1) and von Willebrand Factor (VWF) in infected LoCs at 1 and 3 dpi (n=3 biological replicates in each case) vs. uninfected controls (n=3 biological replicates). Data information: In all plots, bars represent the mean value and error bars represent the standard deviation. Scale bar = 20 μm. P-values are calculated using a one-way Kruskal-Wallis ANOVA test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001.


( A - H ) 3D views of 232 x 232 μm2 fields of view of the AT ( A , G ) and endothelial layer ( B , H ) from infected LoCs reconstituted without macrophages at 1 ( A , B ) and 3 dpi ( G , H ) . Orf1ab antisense RNA , S RNA , ACE2 mRNA , and nuclear staining with DAPI are false - colored pink , amber , spring green , and electric indigo , respectively . ( C , D ) Zooms corresponding to the region in ( A ) highlighted with white and yellow boxes , respectively . ( C ) An example of infection and intracellular replication in a cell with no detectable ACE2 expression ( white arrow ) as well as nuclear localization of viral RNA ( white arrowhead ) . ( D ) An example of an uninfected cell with ACE2 mRNA expression ( yellow arrow ) . ( E , F ) Zooms corresponding to the regions in ( B ) representing increased nucleic acid staining ( hyperplasic ) and normal levels of nucleic acid staining highlighted with white and yellow boxes , respectively . ( E ) Examples of infection of hyperplasic endothelial cells both with and without ACE2 expression ( F ) Examples of endothelial cell infection with no ACE2 expression , nuclear localization of viral RNA in an infected endothelial cell is indicated ( white arrow ) .

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(A-H) 3D views of 232 x 232 μm2 fields of view of the AT (A, G) and endothelial layer (B, H) from infected LoCs reconstituted without macrophages at 1 (A, B) and 3 dpi (G, H). Orf1ab antisense RNA, S RNA, ACE2 mRNA, and nuclear staining with DAPI are false-colored pink, amber, spring green, and electric indigo, respectively. (C, D) Zooms corresponding to the region in (A) highlighted with white and yellow boxes, respectively. (C) An example of infection and intracellular replication in a cell with no detectable ACE2 expression (white arrow) as well as nuclear localization of viral RNA (white arrowhead). (D) An example of an uninfected cell with ACE2 mRNA expression (yellow arrow). (E, F) Zooms corresponding to the regions in (B) representing increased nucleic acid staining (hyperplasic) and normal levels of nucleic acid staining highlighted with white and yellow boxes, respectively. (E) Examples of infection of hyperplasic endothelial cells both with and without ACE2 expression (F) Examples of endothelial cell infection with no ACE2 expression, nuclear localization of viral RNA in an infected endothelial cell is indicated (white arrow).


( A ) Expression relative to GAPDH of pro - inflammatory cytokines ( TNFA , IL1B , IL6 ) , interferon genes ( IFNB , IFNL1 , IFNL3 ) and interferon stimulating genes ( IP10 , ISG15 ) in the AT and endothelial layer of infected LoCs at 1 dpi reconstituted without macrophages ( n = 3 biological replicates for IFNB , IFNL1 and IFNL3 in the AT layer , n = 4 biological replicates for all other data ) . ( B ) Fold - change in the markers in ( A ) relative to uninfected controls ( n = 3 biological replicates ) ( data in Appendix Fig . S12 ) at the same timepoint . The bar represents the mean , and the error bars the standard deviation . ( Data information : The bar represents the mean , and the error bars the standard deviation . P - values are calculated using a Kruskal - Wallis One - Way ANOVA Test , * represents p ≤ 0 . 05 , * * represents p ≤ 0 . 01 , * * represents p ≤ 0 . 001 .

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(A) Expression relative to GAPDH of pro-inflammatory cytokines (TNFA, IL1B, IL6), interferon genes (IFNB, IFNL1, IFNL3) and interferon stimulating genes (IP10, ISG15) in the AT and endothelial layer of infected LoCs at 1 dpi reconstituted without macrophages (n=3 biological replicates for IFNB, IFNL1 and IFNL3 in the AT layer, n=4 biological replicates for all other data). (B) Fold-change in the markers in (A) relative to uninfected controls (n=3 biological replicates) (data in Appendix Fig. S12) at the same timepoint. The bar represents the mean, and the error bars the standard deviation. ( Data information: The bar represents the mean, and the error bars the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001.


( D ) Expression of IL6R and ADAM17 relative to GAPDH in the AT and endothelial layer of infected LoCs at 1 dpi ( n = 3 biological replicates for the AT layer and n = 4 biological replicates for endothelial layer ) . ( E ) Fold - change in expression of IL6 and ADAM17 in the epithelial and endothelial layers of infected LoCs at 1 dpi relative to uninfected controls ( n = 3 biological replicates for both layers ) . ( Data information : The bar represents the mean , and the error bars the standard deviation . P - values are calculated using a Kruskal - Wallis One - Way ANOVA Test , * represents p ≤ 0 . 05 , * * represents p ≤ 0 . 01 , * * represents p ≤ 0 . 001 .

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

(D) Expression of IL6R and ADAM17 relative to GAPDH in the AT and endothelial layer of infected LoCs at 1 dpi (n=3 biological replicates for the AT layer and n=4 biological replicates for endothelial layer). (E) Fold-change in expression of IL6 and ADAM17 in the epithelial and endothelial layers of infected LoCs at 1 dpi relative to uninfected controls (n=3 biological replicates for both layers). ( Data information: The bar represents the mean, and the error bars the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001.


( F - I ) 3D views of representative 232 x 232 μm2 fields of view of the AT ( F , H ) and endothelial layer ( G , I ) from infected LoCs reconstituted with macrophages at 1 ( F , G ) and 3 dpi ( H , I ) . IL6 mRNA , and nuclear staining are indicated with the chartreuse and electric indigo LUTs , respectively .

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(F-I) 3D views of representative 232 x 232 μm2 fields of view of the AT (F, H) and endothelial layer (G, I) from infected LoCs reconstituted with macrophages at 1 (F, G) and 3 dpi (H, I). IL6 mRNA, and nuclear staining are indicated with the chartreuse and electric indigo LUTs, respectively.


( A - F ) Maximum intensity projections of representative 232 x 232 μm2 fields of view of the endothelial layer from an infected LoC reconstituted without macrophages at 2 dpi with ( A ) and without Tocilizumab treatment ( D ) . Actin and nuclear staining are indicated with the azure and electric indigo LUTs respectively , the actin staining is saturated for the endothelial cell clusters to highlight cells with lower levels of actin . ( B , C ) Zooms corresponding to the region in ( A ) highlighted with white and yellow boxes , respectively . ( B ) A region with normal vascular profile , ( C ) A region of endothelial cell cluster formation with increased actin staining . ( E , F ) Zooms corresponding to the regions in ( D ) representing regions highlighted with white and yellow boxes , respectively . ( E ) An example of reduced tight junctions between endothelial cells ; bare patches are indicated with arrows . ( F ) A region of endothelial cell clusters . Scale bar = 20 μm .

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(A-F) Maximum intensity projections of representative 232 x 232 μm2 fields of view of the endothelial layer from an infected LoC reconstituted without macrophages at 2 dpi with (A) and without Tocilizumab treatment (D). Actin and nuclear staining are indicated with the azure and electric indigo LUTs respectively, the actin staining is saturated for the endothelial cell clusters to highlight cells with lower levels of actin. (B, C) Zooms corresponding to the region in (A) highlighted with white and yellow boxes, respectively. (B) A region with normal vascular profile, (C) A region of endothelial cell cluster formation with increased actin staining. (E, F) Zooms corresponding to the regions in (D) representing regions highlighted with white and yellow boxes, respectively. (E) An example of reduced tight junctions between endothelial cells; bare patches are indicated with arrows. (F) A region of endothelial cell clusters. Scale bar = 20 μm.


( G , H ) 3D views from two 232 x 232 μm2 field of views of the endothelial layer of a Tocilizumab treated infected LoC at 2 dpi , immunostaining for the tight junction protein CD31 is shown in the Spring Green LUT . Endothelial cell clusters with low CD31 expression and altered actin staining are also visible and indicated by yellow arrows .

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(G, H) 3D views from two 232 x 232 μm2 field of views of the endothelial layer of a Tocilizumab treated infected LoC at 2 dpi, immunostaining for the tight junction protein CD31 is shown in the Spring Green LUT. Endothelial cell clusters with low CD31 expression and altered actin staining are also visible and indicated by yellow arrows.


B . IHLs were isolated from the liver of Tie2 - GFP mice intrasplenically injected with NaCl ( n = 3 ) or with 5x103 CT26 at the specified time points ( day 25 post - injection n = 6 , day 35 post - injection n = 5 ) . The number of GFP + TEMs was estimated by flow cytometry as 7AAD - / CD45 + / CD11b + / CD11c - / GFP + cells per liver ; data pooled from three independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test .

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B. IHLs were isolated from the liver of Tie2-GFP mice intrasplenically injected with NaCl (n=3) or with 5x103 CT26 at the specified time points (day 25 post-injection n=6, day 35 post-injection n=5). The number of GFP+ TEMs was estimated by flow cytometry as 7AAD-/CD45+/CD11b+/CD11c-/GFP+ cells per liver; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.


C . Representative images ( top panels ) and corresponding H & amp ; amp ; E micrographs ( bottom panels ) of the liver from control ( NaCl - injected , left panels ) or Tie2 - GFP mice 25 and 35 days ( center and right panels , respectively ) post - intrasplenic injection of 5x103 CT26 . Large metastatic foci in the liver of mice that received CT26 cells are indicated ( arrowheads ) . The dashed line in the H & amp ; amp ; E panels identifies the metastasis margin ; ✱ = CRC metastatic area ; scale bars = 100μm .

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C. Representative images (top panels) and corresponding H&amp;E micrographs (bottom panels) of the liver from control (NaCl-injected, left panels) or Tie2-GFP mice 25 and 35 days (center and right panels, respectively) post-intrasplenic injection of 5x103 CT26. Large metastatic foci in the liver of mice that received CT26 cells are indicated (arrowheads). The dashed line in the H&amp;E panels identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm.


D . Liver RNA was extracted from mice transplanted and injected as described in B ( NaCl n = 4 , day 25 n = 7 , day 35 n = 4 ) . The average GFP expression value of NaCl injected mice estimated by quantitative real - time PCR was set to 1 and utilized as reference to calculate the fold increase values of CT26 injected mice ; data pooled from three independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test .

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D. Liver RNA was extracted from mice transplanted and injected as described in B (NaCl n=4, day 25 n=7, day 35 n=4). The average GFP expression value of NaCl injected mice estimated by quantitative real-time PCR was set to 1 and utilized as reference to calculate the fold increase values of CT26 injected mice; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.


E . Confocal immunofluorescence images of representative liver sections from Tie2 - GFP mice that were injected intrasplenically with either NaCl ( upper panels ) or 5x103 CT26 ( bottom panels , 25 days post - injection ) . Note that TEMs ( identified as GFP + , macrophage mannose receptor [ MMR ] + and F4 / 80 + cells ) gather in the proximity of CRC metastatic foci ( identified by the dashed lines ) ; ✱ = CRC metastatic area ; scale bars = 100μm .

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E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice that were injected intrasplenically with either NaCl (upper panels) or 5x103 CT26 (bottom panels, 25 days post-injection). Note that TEMs (identified as GFP+, macrophage mannose receptor [MMR]+ and F4/80+ cells) gather in the proximity of CRC metastatic foci (identified by the dashed lines); ✱=CRC metastatic area; scale bars=100μm.


A . Contrast - enhanced magnetic resonance imaging ( MRI ) of the liver from representative Tie2 - GFP mice ( red frame ) or Tie2 - IFNα ( blue frame ) that were intrasplenically injected with 5x103 CT26 . Red arrows identify CRC liver metastases of representative z - sections . Tumors were characterized as hypointense and slightly - hyperintense regions in T1 and T2 weighted sequences , respectively . Each panel refers to a single mouse analyzed at different time points ; n . a . = not assessed , refers to a mouse euthanized before the specified time point ; scale bars = 5mm . B . Percentage of mice bearing at least one CRC liver metastasis estimated by MRI analysis . Mice were treated as described in A . Mock / Tie2 - GFP n = 11 , Tie2 - IFNα n = 9 ; the oblique black line pattern within the red columns depicts the percentage of mice euthanized or that died before the indicated time point ; p - values were calculated by Fisher ' s exact test . C . Tumor volume quantification measured by MRI analysis of the same mice described in B . Each symbol corresponds to the same mouse analyzed at different time points ; horizontal bars = mean values ; note that Mock / Tie2 - GFP mice were euthanized or died before day 54 ; p - values were calculated by Mann - Whitney test .

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A. Contrast-enhanced magnetic resonance imaging (MRI) of the liver from representative Tie2-GFP mice (red frame) or Tie2-IFNα (blue frame) that were intrasplenically injected with 5x103 CT26. Red arrows identify CRC liver metastases of representative z-sections. Tumors were characterized as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Each panel refers to a single mouse analyzed at different time points; n.a.=not assessed, refers to a mouse euthanized before the specified time point; scale bars=5mm.B. Percentage of mice bearing at least one CRC liver metastasis estimated by MRI analysis. Mice were treated as described in A. Mock/Tie2-GFP n=11, Tie2-IFNα n=9; the oblique black line pattern within the red columns depicts the percentage of mice euthanized or that died before the indicated time point; p-values were calculated by Fisher's exact test.C. Tumor volume quantification measured by MRI analysis of the same mice described in B. Each symbol corresponds to the same mouse analyzed at different time points; horizontal bars=mean values; note that Mock/Tie2-GFP mice were euthanized or died before day 54; p-values were calculated by Mann-Whitney test.


D , E . Quantitative real - time PCR analyses of the relative expression levels of the interferon inducible genes Oas1 ( D ) and Irf7 ( E ) within brain ( n = 4 , n = 2 ) , kidney ( n = 4 , n = 3 ) and metastatic liver ( n = 4 , n = 7 ) of Tie2 - GFP or Tie2 - IFNα mice euthanized 25 days post - intrasplenic injection of 5x103 CT26 . The basal expression of Oas1 and Irf7 estimated in brain of control mice ( i . e . Tie2 - GFP injected with saline ) , was set to 1 and utilized to calculate the fold increase values depicted . Mean values are shown ; error bars = S . E . M . ; p - value was calculated by Mann - Whitney test .

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D, E. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible genes Oas1 (D) and Irf7 (E) within brain (n=4, n=2), kidney (n=4, n=3) and metastatic liver (n=4, n=7) of Tie2-GFP or Tie2-IFNα mice euthanized 25 days post-intrasplenic injection of 5x103 CT26. The basal expression of Oas1 and Irf7 estimated in brain of control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values depicted. Mean values are shown; error bars=S.E.M.; p-value was calculated by Mann-Whitney test.


F . Eighty - four or 54 days post - NaCl or 5x103 CT26 intrasplenic injection , Sham / CTRL ( n = 5 ) or Tie2 - IFNα mice ( n = 7 ) were infected with LCMV ( Armstrong strain , 200 pfu intraperitoneally injected ) . Eight days post - infection white blood cells were isolated and analyzed for LCMV specific CD8 + T cell response . Left panel : percentage of total CD8 + T cells ; middle panel : percentage of CD8 + / NP118 + T cells ( NP118 = recombinant dimeric H - 2d / Ig fusion protein complexed with the immune - dominant H - 2d - restricted LCMV NP118 - 126 peptide ) ; right panel : percentage of CD8 + / IFNγ + T cells after in vitro stimulation with virus - specific H - 2d - restricted peptide ( NP118 - 126 ) ; data pooled from two independent experiments ; mean values are shown ; error bars = S . E . M . ; differences were not statistically significant ( by unpaired Student ' s t - Test ) .

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F. Eighty-four or 54 days post-NaCl or 5x103 CT26 intrasplenic injection, Sham/CTRL (n=5) or Tie2-IFNα mice (n=7) were infected with LCMV (Armstrong strain, 200 pfu intraperitoneally injected). Eight days post-infection white blood cells were isolated and analyzed for LCMV specific CD8+ T cell response. Left panel: percentage of total CD8+ T cells; middle panel: percentage of CD8+/NP118+ T cells (NP118=recombinant dimeric H-2d/Ig fusion protein complexed with the immune-dominant H-2d-restricted LCMV NP118-126 peptide); right panel: percentage of CD8+/IFNγ+ T cells after in vitro stimulation with virus-specific H-2d-restricted peptide (NP118-126); data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; differences were not statistically significant (by unpaired Student's t-Test).


G . Kaplan - Meier survival curves of the indicated groups of mice described in A . Sham ( n = 3 ) , Mock ( wt ) / Tie2 - GFP ( n = 11 ) ; Tie2 - IFNα ( n = 9 ) ; data pooled from three independent experiments ; p = 0 . 001 by log - rank / Mantel - Cox test . The inset images show representative macroscopic photographs of the metastatic progression in the liver of a control animal ( red frame : Mock / Tie2 - GFP , day 35 post - CT26 injection ) opposed to the lack of lesions in Tie2 - IFNα treated mice at later time points ( blue frame : day 450 post - CT26 injection ) .

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G. Kaplan-Meier survival curves of the indicated groups of mice described in A. Sham (n=3), Mock(wt)/Tie2-GFP (n=11); Tie2-IFNα (n=9); data pooled from three independent experiments; p=0.001 by log-rank/Mantel-Cox test. The inset images show representative macroscopic photographs of the metastatic progression in the liver of a control animal (red frame: Mock/Tie2-GFP, day 35 post-CT26 injection) opposed to the lack of lesions in Tie2-IFNα treated mice at later time points (blue frame: day 450 post-CT26 injection).


A . Quantitative real - time PCR analyses of the relative expression levels of the interferon inducible gene Irf7within the liver of Tie2 - GFP or Tie2 - IFNαmice that were intrasplenically injected with either NaCl ( n = 4 and n = 4 , respectively ) or 5x105CT26 - RFP and euthanized 5 minutes ( n = 9 , n = 5 ) , 3 days ( n = 6 , n = 3 ) and 7 days ( n = 5 , n = 3 ) thereafter . The basal expression of Irf7 estimated in control mice ( i . e . Tie2 - GFP injected with saline ) , was set to 1 and utilized to calculate the fold increase values observed at the following time points post - injection . Data pooled from four independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test . The increase in Irf7 expression levels from the liver of Tie2 - GFPmice was statistically significant ( p = 0 . 004 by one - way Anova test , not reported on graph ) .

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

A. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible gene Irf7within the liver of Tie2-GFP or Tie2-IFNαmice that were intrasplenically injected with either NaCl (n=4 and n=4, respectively) or 5x105CT26-RFP and euthanized 5 minutes (n=9, n=5), 3 days (n=6, n=3) and 7 days (n=5, n=3) thereafter. The basal expression of Irf7 estimated in control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values observed at the following time points post-injection. Data pooled from four independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. The increase in Irf7 expression levels from the liver of Tie2-GFPmice was statistically significant (p=0.004 by one-way Anova test, not reported on graph).


B . Representative RFP immunostaining from the liver of Tie2 - GFP mice ( left panels ) or Tie2 - IFNα mice ( right panels ) at the indicated time points after 5x105 CT26 - RFP intrasplenic injection . Arrowheads highlight single or clustered RFP positive cells ; scale bars = 100μm . C . Immunostaining quantification of hepatic CT26 - RFP arrival ( 5 minutes post - injection : Tie2 - GFP n = 6 , Tie2 - IFNα n = 2 ) and expansion ( day 3 post - injection : Tie2 - GFP n = 6 , Tie2 - IFNα n = 3 ; day 7 post - injection : Tie2 - GFP n = 5 , Tie2 - IFNα n = 3 ) in the liver of Tie2 - GFP mice or Tie2 - IFNα mice that were injected with 5x105 CT26 - RFP as described in B . Data pooled from two independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test . The increased percent of RFP + areas in the liver of Tie2 - GFP mice was statistically significant ( p = 0 . 0009 by one - way Anova test , not reported on graph ) .

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B. Representative RFP immunostaining from the liver of Tie2-GFP mice (left panels) or Tie2-IFNα mice (right panels) at the indicated time points after 5x105 CT26-RFP intrasplenic injection. Arrowheads highlight single or clustered RFP positive cells; scale bars=100μm.C. Immunostaining quantification of hepatic CT26-RFP arrival (5 minutes post-injection: Tie2-GFP n=6, Tie2-IFNα n=2) and expansion (day 3 post-injection: Tie2-GFP n=6, Tie2-IFNα n=3; day 7 post-injection: Tie2-GFP n=5, Tie2-IFNα n=3) in the liver of Tie2-GFP mice or Tie2-IFNα mice that were injected with 5x105 CT26-RFP as described in B. Data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. The increased percent of RFP+ areas in the liver of Tie2-GFP mice was statistically significant (p=0.0009 by one-way Anova test, not reported on graph).


A . Representative contrast - enhanced MRI panels depicting the liver of Mock / Tie2 - GFP ( red frame ) or Tie2 - IFNα ( blue frame ) chimeric mice . Each frame shows the metastatic progression 14 and 21 days post - intrasplenic injection of 5x104 MC38 within the indicated chimeric group . BM donors and recipient mouse strains are indicated on the left of each frame ; red arrows or dashed red lines identify CRC liver metastases of representative z - sections . Tumors were identified as hypointense and slightly - hyperintense regions in T1 and T2 weighted sequences respectively ; scale bars = 5mm . B . Tumor volume quantification ( based on MRI analyses ) of lesions detected in the same livers described in A . Chimeric groups and number of mice analyzed are listed ; data pooled from three independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test .

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A. Representative contrast-enhanced MRI panels depicting the liver of Mock/Tie2-GFP (red frame) or Tie2-IFNα (blue frame) chimeric mice. Each frame shows the metastatic progression 14 and 21 days post-intrasplenic injection of 5x104 MC38 within the indicated chimeric group. BM donors and recipient mouse strains are indicated on the left of each frame; red arrows or dashed red lines identify CRC liver metastases of representative z-sections. Tumors were identified as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences respectively; scale bars=5mm.B. Tumor volume quantification (based on MRI analyses) of lesions detected in the same livers described in A. Chimeric groups and number of mice analyzed are listed; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.


B . Tumor volume quantification measured by MRI analysis of Tie2 - GFP ( n = 6 ) or Tie2 - IFNα mice ( n = 9 ) at the indicated time points post - transplant , one additional Tie2 - IFNα mouse showed a tumor volume at day 21 of more than 820 mm3 and was statistically rejected by the Grubbs ' test and not further analyzed ; mean values are shown ; error bars = S . E . M . ; p = 0 . 051 , by Mann - Whitney test .

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B. Tumor volume quantification measured by MRI analysis of Tie2-GFP (n=6) or Tie2-IFNα mice (n=9) at the indicated time points post-transplant, one additional Tie2-IFNα mouse showed a tumor volume at day 21 of more than 820 mm3 and was statistically rejected by the Grubbs' test and not further analyzed; mean values are shown; error bars=S.E.M.; p=0.051, by Mann-Whitney test.


C . Images ( top panels ) and corresponding contrast - enhanced MRI ( middle and bottom panels ) of the liver from representative Tie2 - GFP mice or Tie2 - IFNα mice that were intrahepatically injected with 5x103 CT26 . White dashed lines identify macroscopic lesions . Red and green dashed lines identify hepatic or extrahepatic CRC metastases respectively from representative MRI z - stacks . Tumors detected by MRI analysis appeared as hypointense and slightly - hyperintense regions in T1 and T2 weighted sequences , respectively . Scale bars = 5mm .

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C. Images (top panels) and corresponding contrast-enhanced MRI (middle and bottom panels) of the liver from representative Tie2-GFP mice or Tie2-IFNα mice that were intrahepatically injected with 5x103 CT26. White dashed lines identify macroscopic lesions. Red and green dashed lines identify hepatic or extrahepatic CRC metastases respectively from representative MRI z-stacks. Tumors detected by MRI analysis appeared as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Scale bars=5mm.


D . Percentage of mice with peritoneal carcinomatosis ( defined by the presence of multiple intra - peritoneal lesions ) determined by MRI analysis from the same mice described in B . Note that while intrahepatic number of lesions remained similar between time points , extrahepatic tumor spreading was significantly reduced in Tie2 - IFNα mice . P - values were calculated by Fisher ' s exact test .

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D. Percentage of mice with peritoneal carcinomatosis (defined by the presence of multiple intra-peritoneal lesions) determined by MRI analysis from the same mice described in B. Note that while intrahepatic number of lesions remained similar between time points, extrahepatic tumor spreading was significantly reduced in Tie2-IFNα mice. P-values were calculated by Fisher's exact test.


E . Confocal immunofluorescence images of representative liver sections from Tie2 - GFP mice at distal ( upper panels ) or proximal ( bottom panels ) areas to CRC liver metastasis , 30 days post HSPC transplant . Note that TEMs were identified as GFP + cells and by the concomitant expression of different levels of the myeloid cell marker CD11b + as highlighted in the inset ( merge panels , white arrows ) ; the dashed line identifies the metastasis margin ; ✱ = CRC metastatic area ; scale bars = 100μm ; insets depict the corresponding areas identified by dashed squares in the merge panels , magnified 1 . 5 - folds .

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E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice at distal (upper panels) or proximal (bottom panels) areas to CRC liver metastasis, 30 days post HSPC transplant. Note that TEMs were identified as GFP+ cells and by the concomitant expression of different levels of the myeloid cell marker CD11b+ as highlighted in the inset (merge panels, white arrows); the dashed line identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm; insets depict the corresponding areas identified by dashed squares in the merge panels, magnified 1.5-folds.


F , G . Quantitative real - time PCR analyses of the relative expression levels of the interferon inducible genes Irf7 and Oas1 within liver of Tie2 - GFP ( n = 4 ) or Tie2 - IFNα ( n = 4 ) mice euthanized 30 days post - transplant . The basal expression of Irf7 estimated in liver of control mice ( i . e . Tie2 - GFP injected with saline ) , was set to 1 and utilized to calculate the fold increase values observed . Mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test .

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F, G. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible genes Irf7 and Oas1 within liver of Tie2-GFP (n=4) or Tie2-IFNα (n=4) mice euthanized 30 days post-transplant. The basal expression of Irf7 estimated in liver of control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values observed. Mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.


H . Kaplan - Meier survival curves of the indicated groups of mice . Tie2 - GFP ( n = 3 ) ; Tie2 - IFNα ( n = 5 ) ; p = 0 . 007 by log - rank / Mantel - Cox test .

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H. Kaplan-Meier survival curves of the indicated groups of mice. Tie2-GFP (n=3); Tie2-IFNα (n=5); p=0.007 by log-rank/Mantel-Cox test.


A . IHL and TEM characterization in the liver of patients described in Appendix Fig S6A . Liver samples from surgical resections ( n = 9 ) were weighted and IHLs were isolated , counted and normalized over tissue weight ( left panel ) . Flow cytometry analysis revealed that TEMs , identified as 7AAD - / CD45 + / CD11b + / CD14 + / Tie2 + cells , are detectable within the total IHL populations and accumulate preferentially in the proximity of the metastatic lesions ( right panel ) . Control = hepatic hemangioma ; Distal = distal to CRC liver metastasis ( > 1 cm from the lesion ) ; Proximal = proximal to CRC liver metastasis ( < 1 cm from the lesion ) ; p - values were calculated by Wilcoxon matched pairs Test .

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A. IHL and TEM characterization in the liver of patients described in Appendix Fig S6A. Liver samples from surgical resections (n=9) were weighted and IHLs were isolated, counted and normalized over tissue weight (left panel). Flow cytometry analysis revealed that TEMs, identified as 7AAD-/CD45+/CD11b+/CD14+/Tie2+ cells, are detectable within the total IHL populations and accumulate preferentially in the proximity of the metastatic lesions (right panel). Control=hepatic hemangioma; Distal=distal to CRC liver metastasis (>1 cm from the lesion); Proximal=proximal to CRC liver metastasis (<1 cm from the lesion); p-values were calculated by Wilcoxon matched pairs Test.


B - C . Representative TIE2 immunohistochemistry staining from the liver of a patient with liver CRC metastases from distal ( > 1 cm ; left panels ) or proximal ( < 1 cm ; right panels ) sites from the CRC liver lesion . C . Higher magnification of the panels identified by a rectangle in panel B . Negative control ( bottom panels ) is obtained by omitting primary anti - TIE2 antibodies . TIE2immunostaining , indicates that sites that are distal from the lesion contain only TIE2 + cells with an apparent endothelial morphology ( characterized by an elongated appearance , arrows ) while sites that are proximal to the lesion also contain additional TIE2 + cells with an apparent monocyte - like morphology ( characterized by a round appearance , arrowheads ) . The dashed lines identifies the metastasis margin ; ✱ = CRC metastatic area . Scale bars of upper panels = 50μm ; middle and bottom panels = 20 μm .

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B-C. Representative TIE2 immunohistochemistry staining from the liver of a patient with liver CRC metastases from distal (>1 cm; left panels) or proximal (<1 cm; right panels) sites from the CRC liver lesion. C. Higher magnification of the panels identified by a rectangle in panel B. Negative control (bottom panels) is obtained by omitting primary anti-TIE2 antibodies. TIE2immunostaining, indicates that sites that are distal from the lesion contain only TIE2+ cells with an apparent endothelial morphology (characterized by an elongated appearance, arrows) while sites that are proximal to the lesion also contain additional TIE2+ cells with an apparent monocyte-like morphology (characterized by a round appearance, arrowheads). The dashed lines identifies the metastasis margin; ✱=CRC metastatic area. Scale bars of upper panels=50μm; middle and bottom panels=20 μm.


Cell height was measured using AFM for HeLa - FUCCI in the indicated phase of mitosis . Below : representative images of synchronised HeLa -

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Cell height was measured using AFM for HeLa-FUCCI in the indicated phase of mitosis. Below: representative images of synchronised HeLa-FUCCI


Representative images of synchronised HeLa stained with an anti - geminin to confirm the indicated phase of mitosis ( scale bar 15 µm ) .

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Representative images of synchronised HeLa stained with an anti-geminin to confirm the indicated phase of mitosis (scale bar 15 µm).


Nucleus height was measured by immunofluorescence using Hoechst staining


Representative images of synchronised MEFs stained with an anti - geminin to confirm the indicated phase of

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Representative images of synchronised MEFs stained with an anti-geminin to confirm the indicated phase of mitosis


Nucleus height of synchronised MEFs ( black dot ) and synchronised MRC - 5 ( blue dot ) were measured by immunofluorescence using Hoechst staining . Data are presented as mean ± s . e . m .

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Nucleus height of synchronised MEFs (black dot) and synchronised MRC-5 (blue dot) were measured by immunofluorescence using Hoechst staining. Data are presented as mean ± s.e.m.


Nucleus height was measured by immunofluorescence using lamin A / C staining .

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Nucleus height was measured by immunofluorescence using lamin A/C staining.


Representative HeLa - FUCCI cells in S - G2 - M ( green ) and G1 phase ( red ) in Ctrl , Blebb and Blebb + AP conditions . Scale bar = 31 µm .

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Representative HeLa-FUCCI cells in S-G2-M (green) and G1 phase (red) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 31 µm.


Corresponding percentage of HeLa - FUCCI in mitosis .

Corresponding percentage of HeLa-FUCCI in mitosis.


Representative flow cytometry histograms for EdU positive cells in Ctrl , Blebb and Blebb + AP conditions . Corresponding percentage of EdU positive cells .

Representative flow cytometry histograms for EdU positive cells in Ctrl, Blebb and Blebb + AP conditions. Corresponding percentage of EdU positive cells.


Analysis of transcription factor activity ( Affymetrix combo protein - DNA array ) for Ctrl , Blebb and Blebb + AP conditions . Each square corresponds to a specific transcription factor ( TF ) activity and is color coded to show the relative activity . ① To identify TFs regulated by nuclear shape , we selected TFs that were more active in Ctrl and Blebb + AP compared to blebbistation - treated cells ( Blebb ) .

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Analysis of transcription factor activity (Affymetrix combo protein-DNA array) for Ctrl, Blebb and Blebb + AP conditions. Each square corresponds to a specific transcription factor (TF) activity and is color coded to show the relative activity. ① To identify TFs regulated by nuclear shape, we selected TFs that were more active in Ctrl and Blebb + AP compared to blebbistation-treated cells (Blebb).


TF ' s target genes and GAPDH mRNA levels were analyzed by real - time qPCR . Results are expressed as relative mRNA expression levels .

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TF's target genes and GAPDH mRNA levels were analyzed by real-time qPCR. Results are expressed as relative mRNA expression levels.


TEAD activity was analyzed in HeLa cells co - transfected with a renilla plasmid as a luciferase reporter plasmid controlled by the TEAD - responsive promoter , and with a renilla plasmid as a gene reporter ; HeLa cells were cultured on 1 and 50 kPa ( n = 3 , t - test * * * p < 0 . 01 ) or treated with blebbistatin and blebbistatin + AP ( n = 6 ; t - test * p < 0 . 5 ) .

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TEAD activity was analyzed in HeLa cells co-transfected with a renilla plasmid as a luciferase reporter plasmid controlled by the TEAD-responsive promoter, and with a renilla plasmid as a gene reporter; HeLa cells were cultured on 1 and 50 kPa (n = 3, t-test ***p<0.01) or treated with blebbistatin and blebbistatin + AP (n = 6; t-test *p<0.5).


AP1 activity was assessed in the indicated conditions using AP1 reporter assay .

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AP1 activity was assessed in the indicated conditions using AP1 reporter assay.


Heatmap of mRNA expression profiles of genes regulated by TEAD and AP1 .

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Heatmap of mRNA expression profiles of genes regulated by TEAD and AP1.


Representative cells stained for p - Jun Ser63 ( magenta ) and for DNA ( cyan ) in Ctrl , Blebb and Blebb + AP conditions . Scale bar = 10 µm . Nuclear heights were measured using Hoechst staining . ( b ) Corresponding quantifications of p - Jun Ser63 nuclear intensity .

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Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. (b) Corresponding quantifications of p-Jun Ser63 nuclear intensity.


Representative cells stained for YAP ( magenta ) and for DNA ( cyan ) in Ctrl , Blebb and Blebb + AP conditions . Scale bar = 10 µm . Nuclear heights were measured using Hoechst staining . Corresponding quantifications of YAP activity ( ratio nucleus / cytosol ) .

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Representative cells stained for YAP (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (ratio nucleus/cytosol).


Quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity in Ctrl , Blebb , Blebb + AP condition and in Ctrl , Y27632 and Y27632 + AP .

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Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Blebb, Blebb + AP condition and in Ctrl, Y27632 and Y27632 + AP.


Quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity in Ctrl , Cytochalasin D ( CytoD ) , CytoD + AP condition .

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Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Cytochalasin D (CytoD), CytoD + AP condition.


Quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity in cells depleted or not for SUN1 and SUN2 .

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Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in cells depleted or not for SUN1 and SUN2.


Representative cells cultured on circular micropatterns with surfaces of 1500 and 500 µm2 and stained for p - Jun Ser63 ( magenta ) , YAP ( cyan ) and for DNA ( green ) . Additionally , cells were cultured on the micropatterns of 500 µm2 and an agarose pad was used to flatten their nuclei . Scale bar = 10 µm . Nuclear heights were measured using Hoechst staining . Corresponding quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity .

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Representative cells cultured on circular micropatterns with surfaces of 1500 and 500 µm2 and stained for p-Jun Ser63 (magenta), YAP (cyan) and for DNA (green). Additionally, cells were cultured on the micropatterns of 500 µm2 and an agarose pad was used to flatten their nuclei. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity.


Quantifications of YAP activity in Ctrl , Blebb and Blebb + AP conditions , treated or not with importazol ( nucleo - cytoplasmic ratio ) .

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Quantifications of YAP activity in Ctrl, Blebb and Blebb + AP conditions, treated or not with importazol (nucleo-cytoplasmic ratio).


Immunoblots of p - YAP ( ser127 ) , YAP , p - LATS ( Thr1079 ) , LATS , p - MST ( Thr1083 ) , MST , Kibra , PTPN14 and GADPH for Ctrl , Blebb and Blebb + AP conditions .

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Immunoblots of p-YAP (ser127), YAP, p-LATS (Thr1079), LATS, p-MST (Thr1083), MST, Kibra, PTPN14 and GADPH for Ctrl, Blebb and Blebb + AP conditions.


Corresponding quantification of p - YAP ( Ser125 ) relative to Ctrl and normalized to GAPDH . Corresponding quantification of p - LATS ( thr1079 ) relative to Ctrl and normalized to GAPDH .

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Corresponding quantification of p-YAP (Ser125) relative to Ctrl and normalized to GAPDH. Corresponding quantification of p-LATS (thr1079) relative to Ctrl and normalized to GAPDH.


Quantifications of p - Jun Ser63 nuclear intensity in Ctrl , Blebb , and Blebb + AP conditions for cells treated with DMSO , SB203580 , a MAP Kinase inhibitor and SP600125 , a JNK inhibitor .

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Quantifications of p-Jun Ser63 nuclear intensity in Ctrl, Blebb, and Blebb + AP conditions for cells treated with DMSO, SB203580, a MAP Kinase inhibitor and SP600125, a JNK inhibitor.


Representative cells stained for p - Jun Ser63 ( magenta ) and for DNA ( cyan ) in Ctrl , Blebb and Blebb + AP conditions for cells depleted or not for lamin - A / C . Scale bar = 10 µm . Corresponding quantification of p - Jun Ser63 nuclear intensity .

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Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions for cells depleted or not for lamin-A/C. Scale bar = 10 µm. Corresponding quantification of p-Jun Ser63 nuclear intensity.


A ) Representative immunofluorescence images showing localization of Myc - tagged M18BP1 - 1 truncations schematized in ( B ) to metaphase sperm centromeres in M18BP1 - depleted extract . Truncations analyzed are indicated on the left , immunolocalized proteins are indicated above . Scale bar , 10 μm . Insets are magnified 3X . B ) Schematic of M18BP1 - 1 truncations used to define the metaphase targeting domain in ( A ) and ( C ) . C ) Quantification of the average Myc intensity at centromeres from ( A ) . Values are normalized to centromeric signal of full - length M18BP1 - 1 . Graph shows the mean ± SEM of at least three experiments . Significance determined by Welch ' s unpaired two - tailed t - test , * p < 0 . 05 , * * p < 0 . 005 .

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A) Representative immunofluorescence images showing localization of Myc-tagged M18BP1-1 truncations schematized in (B) to metaphase sperm centromeres in M18BP1-depleted extract. Truncations analyzed are indicated on the left, immunolocalized proteins are indicated above. Scale bar, 10 μm. Insets are magnified 3X. B) Schematic of M18BP1-1 truncations used to define the metaphase targeting domain in (A) and (C). C) Quantification of the average Myc intensity at centromeres from (A). Values are normalized to centromeric signal of full-length M18BP1-1. Graph shows the mean ± SEM of at least three experiments. Significance determined by Welch's unpaired two-tailed t-test, * p<0.05, ** p<0.005.


B ) Representative Western blot showing disruption of the interaction between M18BP1 - 1 and CENP - C by mutation of the SANTA domain by co - immunoprecipitation . Extract depleted of endogenous M18BP1 was supplemented with full - length Flag - M18BP1 - 1WT or Flag - M18BP1 - 1SANTA . The M18BP1 - 1 species added to each reaction and the cell cycle state of the extract is indicated above . The immunoblotted species is indicated at left . Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control .

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B) Representative Western blot showing disruption of the interaction between M18BP1-1 and CENP-C by mutation of the SANTA domain by co-immunoprecipitation. Extract depleted of endogenous M18BP1 was supplemented with full-length Flag-M18BP1-1WT or Flag-M18BP1-1SANTA. The M18BP1-1 species added to each reaction and the cell cycle state of the extract is indicated above. The immunoblotted species is indicated at left. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control.


C ) Representative images showing that mutation of the SANTA domain in M18BP1 - 1 causes decreased localization to sperm centromeres . Metaphase extracts were depleted of endogenous M18BP1 and either mock - depleted or depleted of CENP - C . The extracts were then complemented with WT or SANTA mutant M18BP1 - 1 . M18BP1 - 1 species and depletion conditions are indicated at left , immunolocalized proteins are indicated above . Scale bar , 10 μm . Insets are magnified 3X . D ) Quantification of the average Flag - M18BP1 - 1 intensity at centromeres from ( C ) . Values are normalized to centromeric signal of M18BP1 - 1WT in mock - depleted extract . Graph shows the mean ± SEM of three experiments . Significance determined by Welch ' s unpaired two - tailed t - test , * * p < 0 . 005 .

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C) Representative images showing that mutation of the SANTA domain in M18BP1-1 causes decreased localization to sperm centromeres. Metaphase extracts were depleted of endogenous M18BP1 and either mock-depleted or depleted of CENP-C. The extracts were then complemented with WT or SANTA mutant M18BP1-1. M18BP1-1 species and depletion conditions are indicated at left, immunolocalized proteins are indicated above. Scale bar, 10 μm. Insets are magnified 3X. D) Quantification of the average Flag-M18BP1-1 intensity at centromeres from (C). Values are normalized to centromeric signal of M18BP1-1WT in mock-depleted extract. Graph shows the mean ± SEM of three experiments. Significance determined by Welch's unpaired two-tailed t-test, ** p<0.005.


A ) A representative autoradiograph from a GST - pulldown of CENP - C to map the CENP - C binding domain of M18BP1 - 1 . Radiolabeled Myc - M18BP1 - 1 truncations ( amino acids indicated at the top ) were mixed with recombinant GST - CENP - C1191 - 1400 . Material bound to glutathione agarose was resolved by SDS - PAGE and visualized by autoradiography C ) Quantification of ( A ) . Bound material as a fraction of the input was calculated from autoradiographs . The graph shows mean fraction bound ± SD of three independent experiments normalized to M18BP1 - 1161 - 580 , the CENP - C binding domain .

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A) A representative autoradiograph from a GST-pulldown of CENP-C to map the CENP-C binding domain of M18BP1-1. Radiolabeled Myc-M18BP1-1 truncations (amino acids indicated at the top) were mixed with recombinant GST-CENP-C1191-1400. Material bound to glutathione agarose was resolved by SDS-PAGE and visualized by autoradiography C) Quantification of (A). Bound material as a fraction of the input was calculated from autoradiographs. The graph shows mean fraction bound ± SD of three independent experiments normalized to M18BP1-1161-580, the CENP-C binding domain.


D ) Representative Western blot showing metaphase - specific binding of M18BP1 - 1161 - 580 to CENP - C by co - immunoprecipitation . Extract depleted of endogenous M18BP1 was supplemented with full - length Myc - M18BP1 - 1 or Myc - M18BP1 - 1161 - 580 . M18BP1 - 1 species added to each reaction and cell cycle state of the extract is indicated above . Immunoblotted species indicated at left . Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control .

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D) Representative Western blot showing metaphase-specific binding of M18BP1-1161-580 to CENP-C by co-immunoprecipitation. Extract depleted of endogenous M18BP1 was supplemented with full-length Myc-M18BP1-1 or Myc-M18BP1-1161-580. M18BP1-1 species added to each reaction and cell cycle state of the extract is indicated above. Immunoblotted species indicated at left. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control.


E ) Representative immunofluorescence images showing Myc - M18BP1 - 1161 - 580 localization at sperm centromeres in extract depleted of endogenous M18BP1 . In addition , extract was mock - depleted or immunodepleted of CENP - C ( indicated at left ) . Cell cycle state indicated at left , immunolocalized protein indicated above . Scale bar , 10 μm . Insets magnified 3X . F ) Quantification of ( E ) . Graph shows mean Myc - M18BP1 - 1161 - 580 intensity at centromeres ± SEM of two independent experiments normalized to the mock - depleted , metaphase condition .

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E) Representative immunofluorescence images showing Myc-M18BP1-1161-580 localization at sperm centromeres in extract depleted of endogenous M18BP1. In addition, extract was mock-depleted or immunodepleted of CENP-C (indicated at left). Cell cycle state indicated at left, immunolocalized protein indicated above. Scale bar, 10 μm. Insets magnified 3X. F) Quantification of (E). Graph shows mean Myc-M18BP1-1161-580 intensity at centromeres ± SEM of two independent experiments normalized to the mock-depleted, metaphase condition.


A , B ) Metaphase binding of M18BP1 - 1 to CENP - C requires phosphorylation . Either full - length Myc - M18BP1 - 1 ( A ) or Myc - M18BP1 - 1161 - 580 ( B ) was immunoprecipitated from metaphase extract . Half of the immunoprecipitate was treated with λ - protein phosphatase ( + LPPase ) to assess whether binding to CENP - C required phosphorylation . Immunoprecipitation with an equivalent amount of mouse IgG served as a negative control . Immunoblotted species is indicated at left .

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A, B) Metaphase binding of M18BP1-1 to CENP-C requires phosphorylation. Either full-length Myc-M18BP1-1 (A) or Myc-M18BP1-1161-580 (B) was immunoprecipitated from metaphase extract. Half of the immunoprecipitate was treated with λ-protein phosphatase (+ LPPase) to assess whether binding to CENP-C required phosphorylation. Immunoprecipitation with an equivalent amount of mouse IgG served as a negative control. Immunoblotted species is indicated at left.


C ) Cdk inhibition with flavopiridol prevents M18BP1 - 1161 - 580 localization at metaphase centromeres . Representative images showing Myc - M18BP1 - 1161 - 580 localization at sperm centromeres in metaphase extract depleted of endogenous M18BP1 following treatment with various mitotic kinase inhibitors . Inhibitor treatment is indicated at left , immunolocalized protein is indicated above . Scale bar , 10 μm . Insets are magnified 3X .

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C) Cdk inhibition with flavopiridol prevents M18BP1-1161-580 localization at metaphase centromeres. Representative images showing Myc-M18BP1-1161-580 localization at sperm centromeres in metaphase extract depleted of endogenous M18BP1 following treatment with various mitotic kinase inhibitors. Inhibitor treatment is indicated at left, immunolocalized protein is indicated above. Scale bar, 10 μm. Insets are magnified 3X.


E ) Quantification of immunofluorescence experiments examining localization of M18BP1 - 1161 - 580 phosphorylation site mutants to sperm centromeres in metaphase extract depleted of endogenous M18BP1 . Graph shows mean centromere intensity ± SEM of three independent experiments normalized to the WT condition . Significance determined by Welch ' s unpaired two - tailed t - test , * p < 0 . 05 , * * p < 0 . 005 .

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E) Quantification of immunofluorescence experiments examining localization of M18BP1-1161-580 phosphorylation site mutants to sperm centromeres in metaphase extract depleted of endogenous M18BP1. Graph shows mean centromere intensity ± SEM of three independent experiments normalized to the WT condition. Significance determined by Welch's unpaired two-tailed t-test, * p<0.05, ** p<0.005.


A ) Representative immunofluorescence images showing M18BP1 - 1 mutant localization . M18BP1 - depleted metaphase extract was supplemented with in vitro translated full - length Flag - M18BP1 - 1 mutants and sperm chromatin . In addition , extract was either mock - depleted or CENP - C depleted to assess CENP - C dependent metaphase localization . M18BP1 - 1 species and CENP - C depletion status are indicated at left , immunolocalized protein is indicated above . Scale bar , 10 μm . Insets are magnified 3X .

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A) Representative immunofluorescence images showing M18BP1-1 mutant localization. M18BP1-depleted metaphase extract was supplemented with in vitro translated full-length Flag-M18BP1-1 mutants and sperm chromatin. In addition, extract was either mock-depleted or CENP-C depleted to assess CENP-C dependent metaphase localization. M18BP1-1 species and CENP-C depletion status are indicated at left, immunolocalized protein is indicated above. Scale bar, 10 μm. Insets are magnified 3X.


B ) Representative Western blot showing co - immunoprecipitation of CENP - C with phosphorylation site mutants of M18BP1 - 1 . M18BP1 - depleted metaphase or interphase extract was supplemented with in vitro translated Flag - M18BP1 - 1 mutants . M18BP1 - 1 mutation and cell cycle state indicated above , immunoblotted species indicated at right . Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control .

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B) Representative Western blot showing co-immunoprecipitation of CENP-C with phosphorylation site mutants of M18BP1-1. M18BP1-depleted metaphase or interphase extract was supplemented with in vitro translated Flag-M18BP1-1 mutants. M18BP1-1 mutation and cell cycle state indicated above, immunoblotted species indicated at right. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control.


C ) Quantification of ( A ) . Flag - M18BP1 - 1 mutant centromere intensity normalized to WT localization in the mock - depleted condition . Error bars represent SEM from three independent experiments .

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C) Quantification of (A). Flag-M18BP1-1 mutant centromere intensity normalized to WT localization in the mock-depleted condition. Error bars represent SEM from three independent experiments.


A ) Representative immunofluorescence images showing incorporation of exogenous Myc - CENP - A at centromeres . Sperm nuclei were incubated in M18BP1 - depleted interphase Xenopus egg extracts complemented with the indicated M18BP1 - 1 protein . Extracts were supplemented with RNA encoding Myc - CENP - A to track new CENP - A assembly and in vitro translated HJURP . Scale bar , 10 μm . Insets are magnified 3X . B ) Quantification of Myc - CENP - A loading at Xenopus sperm centromeres in ( A ) . Values are normalized to the centromere signals in mock - depleted extract . Dashed lines indicate the Myc - CENP - A assembly signal observed upon M18BP1 depletion ( bottom ) and Flag - M18BP1 - 1WT add - back ( top ) as points of reference for mutant rescue . Graph shows the mean ± SEM of four independent experiments .

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A) Representative immunofluorescence images showing incorporation of exogenous Myc-CENP-A at centromeres. Sperm nuclei were incubated in M18BP1-depleted interphase Xenopus egg extracts complemented with the indicated M18BP1-1 protein. Extracts were supplemented with RNA encoding Myc-CENP-A to track new CENP-A assembly and in vitro translated HJURP. Scale bar, 10 μm. Insets are magnified 3X. B) Quantification of Myc-CENP-A loading at Xenopus sperm centromeres in (A). Values are normalized to the centromere signals in mock-depleted extract. Dashed lines indicate the Myc-CENP-A assembly signal observed upon M18BP1 depletion (bottom) and Flag-M18BP1-1WT add-back (top) as points of reference for mutant rescue. Graph shows the mean ± SEM of four independent experiments.


C ) Interphase Mis18 complex formation is unaffected by M18BP1 - 1 mutations that prevent binding to CENP - C . Extract depleted of endogenous M18BP1 was supplemented with Myc - Mis18α , Myc - Mis18β , and Flag - M18BP1 - 1 . M18BP1 - 1 species added to each reaction and cell cycle state of the extract is indicated at the top . Co - immunoprecipitation of Myc - Mis18α / β was assessed by α - Myc immunoblot following Flag precipitation .

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C) Interphase Mis18 complex formation is unaffected by M18BP1-1 mutations that prevent binding to CENP-C. Extract depleted of endogenous M18BP1 was supplemented with Myc-Mis18α, Myc-Mis18β, and Flag-M18BP1-1. M18BP1-1 species added to each reaction and cell cycle state of the extract is indicated at the top. Co-immunoprecipitation of Myc-Mis18α/β was assessed by α-Myc immunoblot following Flag precipitation.


D ) HJURP association with M18BP1 - 1 is unaffected by mutations that prevent CENP - C binding . Extract depleted of endogenous M18BP1 was supplemented with Flag - M18BP1 - 1 . M18BP1 - 1 species added to each reaction and cell cycle state of the extract are indicated at the top . Coimmunoprecipitation of Flag - M18BP1 - 1 was assessed by α - Flag immunoblot following HJURP precipitation .

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D) HJURP association with M18BP1-1 is unaffected by mutations that prevent CENP-C binding. Extract depleted of endogenous M18BP1 was supplemented with Flag-M18BP1-1. M18BP1-1 species added to each reaction and cell cycle state of the extract are indicated at the top. Coimmunoprecipitation of Flag-M18BP1-1 was assessed by α-Flag immunoblot following HJURP precipitation.