Figure 2 ( A ) Fluorescence microscopy demonstrates that Lam6 - GFP co - localizes with ERMES ( Mdm34 - Cherry ) ( yellow arrows ) and also localizes to non - ERMES locations in the cell ( red arrows ) . These additional locations co - localized with the NVJ ( Nvj1 - GFP ) as well as with the vCLAMP ( GFP - Vps39 ) . Scale bar represents 5 μm . ( B ) Overexpression and tagging of Lam6 confirmed that it co - localizes with ERMES ( Mdm34 - GFP ) ( yellow arrows ) as well as to additional contact sites ( red arrows ) , NVJ ( marked by Nvj1 ) and vCLAMP ( marked by Vps39 ) . Scale bar represents 5 μm .

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Figure 2(A) Fluorescence microscopy demonstrates that Lam6-GFP co-localizes with ERMES (Mdm34-Cherry) (yellow arrows) and also localizes to non-ERMES locations in the cell (red arrows). These additional locations co-localized with the NVJ (Nvj1-GFP) as well as with the vCLAMP (GFP-Vps39). Scale bar represents 5 μm.(B) Overexpression and tagging of Lam6 confirmed that it co-localizes with ERMES (Mdm34-GFP) (yellow arrows) as well as to additional contact sites (red arrows), NVJ (marked by Nvj1) and vCLAMP (marked by Vps39). Scale bar represents 5 μm.


Figure 3 ( A ) Fluorescence microscopy demonstrates that the overexpression of Cherry - Lam6 ( OE - Cherry - LAM6 ) results in an expansion of the following three contact sites : ERMES ( Mdm34 - GFP ) , NVJ ( Nvj1 - GFP ) , and vCLAMP ( GFP - Vps39 ) . This suggests that an increase in Lam6 levels in the contact site is sufficient for its expansion . The numbers represent the average contact site size ( 120 cells per sample ) . Scale bar represents 5 μm . ( B ) Immuno - EM verified that GFP - Lam6 overexpression indeed causes an expansion of contact site size . While vCLAMP was hardly visible in WT cells , it could be detected easily in the OE strain ( albeit often had ER tubules invading it ) . The NVJ underwent expansion as well as evoked PMN in OE strains , and the ER - mitochondria contact became large and elongated instead of small and distinct . N , nucleus ; M , mitochondria ; V , vacuole . Scale bar represents 200 nm ( see also Figure S3I ) .

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Figure 3(A) Fluorescence microscopy demonstrates that the overexpression of Cherry-Lam6 (OE-Cherry-LAM6) results in an expansion of the following three contact sites: ERMES (Mdm34-GFP), NVJ (Nvj1-GFP), and vCLAMP (GFP-Vps39). This suggests that an increase in Lam6 levels in the contact site is sufficient for its expansion. The numbers represent the average contact site size (120 cells per sample). Scale bar represents 5 μm.(B) Immuno-EM verified that GFP-Lam6 overexpression indeed causes an expansion of contact site size. While vCLAMP was hardly visible in WT cells, it could be detected easily in the OE strain (albeit often had ER tubules invading it). The NVJ underwent expansion as well as evoked PMN in OE strains, and the ER-mitochondria contact became large and elongated instead of small and distinct. N, nucleus; M, mitochondria; V, vacuole. Scale bar represents 200 nm (see also Figure S3I).


Figure 4 ( A ) Fluorescent microscopy shows that the ERMES contact ( as measured by the number of Mdm34 - GFP puncta per cell ) expanded in the Δvps39 background relative to WT . However , the expansion did not occur on the background of Δvps39 Δlam6 , demonstrating that Lam6 is necessary for ERMES expansion under these conditions . Scale bar represents 5 μm . ( B ) Quantitation of ( A ) . Bars represent the percentage of cells containing the specific number of puncta / cell out of total cells counted for the strain ( for WT , n = 234 cells ; for Δvps39 , n = 246 ; for Δvps39 Δlam6 , n = 271 ) . ( C ) Fluorescent microscopy demonstrates that downregulating ERMES contacts ( by growing GALp - MDM34 strains in glucose ) indeed caused expansion of vCLAMP ( GFP - Vps39 ) . However , this expansion was diminished in a Δlam6 background . Scale bar represents 5 μm ( see also Figure S4 ) .

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Figure 4(A) Fluorescent microscopy shows that the ERMES contact (as measured by the number of Mdm34-GFP puncta per cell) expanded in the Δvps39 background relative to WT. However, the expansion did not occur on the background of Δvps39 Δlam6, demonstrating that Lam6 is necessary for ERMES expansion under these conditions. Scale bar represents 5 μm.(B) Quantitation of (A). Bars represent the percentage of cells containing the specific number of puncta/cell out of total cells counted for the strain (for WT, n = 234 cells; for Δvps39, n = 246; for Δvps39 Δlam6, n = 271).(C) Fluorescent microscopy demonstrates that downregulating ERMES contacts (by growing GALp-MDM34 strains in glucose) indeed caused expansion of vCLAMP (GFP-Vps39). However, this expansion was diminished in a Δlam6 background. Scale bar represents 5 μm (see also Figure S4).


Figure 2 ( B ) Autophagy ‐ negative phenotype of the apg16Δ strain . The autophagic ability of wild ‐ type ( TN125 ) , apg12Δ ( YNM107 ) and apg16Δ cells ( YNM114 ) was measured by the alkaline phosphatase assay before ( black bars ) and after ( white bars ) nitrogen starvation for 4 h . Error bars indicate the standard deviation of three independent experiments . ( C ) Loss of viability during starvation . Wild ‐ type ( KA311B , ○ ) , apg12Δ ( YNM101 , ● ) and apg16Δ cells ( YNM124 , ▴ ) were cultured in nitrogen starvation medium , and their viability was determined by phloxine B staining . ( D ) Defect in API maturation in apg16Δ cells . Transport of pro ‐ API to the vacuole was examined by immunoblotting with anti ‐ API antiserum . The positions of precursor and mature API are indicated . ( E ) The Apg12p - Apg5p conjugation in apg16Δ cells . Total lysates were prepared by the NaOH / 2 ‐ mercaptoethanol extraction method from apg12Δ ( YNM107 ) and apg12Δapg16Δ cells ( YNM115 ) , both carrying pHA ‐ APG12 ( CEN ) . Western blot analysis was performed using anti ‐ HA antibody .

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Figure 2(B) Autophagy‐negative phenotype of the apg16Δ strain. The autophagic ability of wild‐type (TN125), apg12Δ (YNM107) and apg16Δ cells (YNM114) was measured by the alkaline phosphatase assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate the standard deviation of three independent experiments.(C) Loss of viability during starvation. Wild‐type (KA311B, ○), apg12Δ (YNM101, ●) and apg16Δ cells (YNM124, ▴) were cultured in nitrogen starvation medium, and their viability was determined by phloxine B staining.(D) Defect in API maturation in apg16Δ cells. Transport of pro‐API to the vacuole was examined by immunoblotting with anti‐API antiserum. The positions of precursor and mature API are indicated.(E) The Apg12p-Apg5p conjugation in apg16Δ cells. Total lysates were prepared by the NaOH/2‐mercaptoethanol extraction method from apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115), both carrying pHA‐APG12 (CEN). Western blot analysis was performed using anti‐HA antibody.


Figure 3Apg16p interacts with the Apg12p - Apg5p conjugate but not with the Apg12p monomer . The apg12Δapg16Δ cells ( YNM115 ) were transformed with high copy ( 2μ ) plasmids encoding MycApg12p and / or HAApg16p as indicated . Total lysates were immunoprecipitated with anti ‐ Myc ( lanes 1 - 4 ) or anti ‐ HA antibody ( lanes 5 - 8 ) and detected by immunoblotting using anti ‐ Myc or anti ‐ HA antibody . The positions of the IgG heavy chain and κ light chain are indicated . Apg16p interacts with the Apg12p - Apg5p conjugate but not with the Apg12p monomer . The apg12Δapg16Δ cells ( YNM115 ) were transformed with high copy ( 2μ ) plasmids encoding MycApg12p and / or HAApg16p as indicated . Total lysates were immunoprecipitated with anti ‐ Myc ( lanes 1 - 4 ) or anti ‐ HA antibody ( lanes 5 - 8 ) and detected by immunoblotting using anti ‐ Myc or anti ‐ HA antibody . The positions of the IgG heavy chain and κ light chain are indicated .

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Figure 3Apg16p interacts with the Apg12p-Apg5p conjugate but not with the Apg12p monomer. The apg12Δapg16Δ cells (YNM115) were transformed with high copy (2μ) plasmids encoding MycApg12p and/or HAApg16p as indicated. Total lysates were immunoprecipitated with anti‐Myc (lanes 1-4) or anti‐HA antibody (lanes 5-8) and detected by immunoblotting using anti‐Myc or anti‐HA antibody. The positions of the IgG heavy chain and κ light chain are indicated.Apg16p interacts with the Apg12p-Apg5p conjugate but not with the Apg12p monomer. The apg12Δapg16Δ cells (YNM115) were transformed with high copy (2μ) plasmids encoding MycApg12p and/or HAApg16p as indicated. Total lysates were immunoprecipitated with anti‐Myc (lanes 1-4) or anti‐HA antibody (lanes 5-8) and detected by immunoblotting using anti‐Myc or anti‐HA antibody. The positions of the IgG heavy chain and κ light chain are indicated.


Figure 4Apg16p interacts preferentially with Apg12p ‐ modified Apg5p . Total lysates were prepared from apg16Δ cells ( YNM124 ) or apg12Δapg16Δ cells ( YNM115 ) harboring both pHA ‐ APG5 and pMyc ‐ APG16 CEN plasmids . The lysates were immunoprecipitated and analyzed as described in Figure 3 . Apg16p interacts preferentially with Apg12p ‐ modified Apg5p . Total lysates were prepared from apg16Δcells ( YNM124 ) or apg12Δapg16Δ cells ( YNM115 ) harboring both pHA ‐ APG5 and pMyc ‐ APG16 CEN plasmids . The lysates were immunoprecipitated and analyzed as described in Figure 3 .

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Figure 4Apg16p interacts preferentially with Apg12p‐modified Apg5p. Total lysates were prepared from apg16Δ cells (YNM124) or apg12Δapg16Δ cells (YNM115) harboring both pHA‐APG5 and pMyc‐APG16 CEN plasmids. The lysates were immunoprecipitated and analyzed as described in Figure 3.Apg16p interacts preferentially with Apg12p‐modified Apg5p. Total lysates were prepared from apg16Δcells (YNM124) or apg12Δapg16Δ cells (YNM115) harboring both pHA‐APG5 and pMyc‐APG16 CEN plasmids. The lysates were immunoprecipitated and analyzed as described in Figure 3.


Figure 5 ( A ) PJ69 ‐ 4A cells were co ‐ transformed with each pGBD and pGAD plasmid as indicated . Transformants were selected on Trp − Leu − plates , and then two ‐ hybrid interaction ( + or − ) was assessed for growth on Ade − Trp − Leu − plates . ( B ) Self ‐ multimerization of Apg16p in vivo . Lysates of apg16Δ cells ( YNM124 ) transformed with pMyc ‐ APG16 ( 2μ ) and / or pHA ‐ APG16 ( 2μ ) were immunoprecipitated with anti ‐ Myc antibody and detected with anti ‐ HA antibody .

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Figure 5(A) PJ69‐4A cells were co‐transformed with each pGBD and pGAD plasmid as indicated. Transformants were selected on Trp− Leu− plates, and then two‐hybrid interaction (+ or −) was assessed for growth on Ade− Trp− Leu− plates.(B) Self‐multimerization of Apg16p in vivo. Lysates of apg16Δ cells (YNM124) transformed with pMyc‐APG16 (2μ) and/or pHA‐APG16 (2μ) were immunoprecipitated with anti‐Myc antibody and detected with anti‐HA antibody.


Figure 6Apg16p mediates Apg5p assembly . Immunoprecipitation was carried out as described in Figure 3 , using apg12Δ ( YNM107 ) and apg12Δapg16Δ cells ( YNM115 ) carrying pMyc ‐ APG12 ( CEN ) and pHA ‐ APG12 ( CEN ) ( A ) , or pMyc ‐ APG12 ( CEN ) and pHA ‐ APG5 ( CEN ) ( B ) . Following SDS - PAGE , immunoprecipitates were subjected to immunoblot analysis using anti ‐ HA or anti ‐ Myc antibody as indicated . A model of the Apg12p - Apg5p - Apg16p complex is illustrated in each panel . Apg16p forms a homo ‐ oligomer through its C ‐ terminal coiled ‐ coil region . It is not clear whether Apg16p forms a dimer or a larger multimer . The N ‐ terminal region of each Apg16p interacts with Apg5p ; most of these complexes are conjugated to Apg12p ( A ) but a small population are unmodified ( B ) . In the absence of Apg16p , separate Apg5ps do not assemble . Apg16p mediates Apg5p assembly . Immunoprecipitation was carried out as described in Figure 3 , using apg12Δ ( YNM107 ) and apg12Δapg16Δ cells ( YNM115 ) carrying pMyc ‐ APG12 ( CEN ) and pHA ‐ APG5 ( CEN ) ( B ) . Following SDS - PAGE , immunoprecipitates were subjected to immunoblot analysis using anti ‐ HA or anti ‐ Myc antibody as indicated . A model of the Apg12p - Apg5p - Apg16p complex is illustrated in each panel . Apg16p forms a homo ‐ oligomer through its C ‐ terminal coiled ‐ coil region . It is not clear whether Apg16p forms a dimer or a larger multimer . The N ‐ terminal region of each Apg16p interacts with Apg5p ; most of these complexes are conjugated to Apg12p ( A ) but a small population are unmodified ( B ) . In the absence of Apg16p , separate Apg5ps do not assemble .

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Figure 6Apg16p mediates Apg5p assembly. Immunoprecipitation was carried out as described in Figure 3, using apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115) carrying pMyc‐APG12 (CEN) and pHA‐APG12 (CEN) (A), or pMyc‐APG12 (CEN) and pHA‐APG5 (CEN) (B). Following SDS-PAGE, immunoprecipitates were subjected to immunoblot analysis using anti‐HA or anti‐Myc antibody as indicated. A model of the Apg12p-Apg5p-Apg16p complex is illustrated in each panel. Apg16p forms a homo‐oligomer through its C‐terminal coiled‐coil region. It is not clear whether Apg16p forms a dimer or a larger multimer. The N‐terminal region of each Apg16p interacts with Apg5p; most of these complexes are conjugated to Apg12p (A) but a small population are unmodified (B). In the absence of Apg16p, separate Apg5ps do not assemble.Apg16p mediates Apg5p assembly. Immunoprecipitation was carried out as described in Figure 3, using apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115) carrying pMyc‐APG12 (CEN) and pHA‐APG5 (CEN) (B). Following SDS-PAGE, immunoprecipitates were subjected to immunoblot analysis using anti‐HA or anti‐Myc antibody as indicated. A model of the Apg12p-Apg5p-Apg16p complex is illustrated in each panel. Apg16p forms a homo‐oligomer through its C‐terminal coiled‐coil region. It is not clear whether Apg16p forms a dimer or a larger multimer. The N‐terminal region of each Apg16p interacts with Apg5p; most of these complexes are conjugated to Apg12p (A) but a small population are unmodified (B). In the absence of Apg16p, separate Apg5ps do not assemble.


Figure 7Expression levels of Apg16p are dependent on the Apg12p - Apg5p conjugate . Total lysates were prepared by the NaOH / 2 ‐ mercaptoethanol extraction method from apg16Δ ( YNM124 ) , apg5Δapg16Δ ( YNM126 ) and apg12Δapg16Δ cells ( YNM115 ) carrying pHA ‐ APG16 ( CEN ) . Western blot analysis was performed using anti ‐ HA antibody . Expression levels of Apg16p are dependent on the Apg12p - Apg5p conjugate . Total lysates were prepared by the NaOH / 2 ‐ mercaptoethanol extraction method from apg16Δ ( YNM124 ) , apg5Δapg16Δ ( YNM126 ) and apg12Δapg16Δ cells ( YNM115 ) carrying pHA ‐ APG16 ( CEN ) . Western blot analysis was performed using anti ‐ HA antibody .

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Figure 7Expression levels of Apg16p are dependent on the Apg12p-Apg5p conjugate. Total lysates were prepared by the NaOH/2‐mercaptoethanol extraction method from apg16Δ (YNM124), apg5Δapg16Δ (YNM126) and apg12Δapg16Δ cells (YNM115) carrying pHA‐APG16 (CEN). Western blot analysis was performed using anti‐HA antibody.Expression levels of Apg16p are dependent on the Apg12p-Apg5p conjugate. Total lysates were prepared by the NaOH/2‐mercaptoethanol extraction method from apg16Δ (YNM124), apg5Δapg16Δ (YNM126) and apg12Δapg16Δ cells (YNM115) carrying pHA‐APG16 (CEN). Western blot analysis was performed using anti‐HA antibody.


Figure 8Apg16p exists in a pellet fraction in an Apg5p ‐ dependent manner . Spheroplasts were generated from cells harboring pHA ‐ APG5 ( A ) , pHA ‐ APG16 ( B ) or pHA ‐ APG16 ( 2μ ) with or without pHA ‐ APG5 ( 2μ ) ( C ) . They were lysed by Dounce homogenization . After unbroken spheroplasts were removed by low ‐ speed centrifugation , the supernatant ( T ) was fractionated by 100 000 g centrifugation for 60 min to generate a supernatant ( S ) and pellet ( P ) fraction . Each fraction was subjected to immunoblot analysis using anti ‐ HA antibody .

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Figure 8Apg16p exists in a pellet fraction in an Apg5p‐dependent manner. Spheroplasts were generated from cells harboring pHA‐APG5 (A), pHA‐APG16 (B) or pHA‐APG16 (2μ) with or without pHA‐APG5 (2μ) (C). They were lysed by Dounce homogenization. After unbroken spheroplasts were removed by low‐speed centrifugation, the supernatant (T) was fractionated by 100 000 g centrifugation for 60 min to generate a supernatant (S) and pellet (P) fraction. Each fraction was subjected to immunoblot analysis using anti‐HA antibody.


Figure 1A ) 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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Figure 1A) 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.


Figure 2B ) 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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Figure 2B) 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.


Figure 3A ) 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 . 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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Figure 3A) 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.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.


Figure 4A , 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 . 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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Figure 4A, 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.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.


Figure 5A ) 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) 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.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.


Figure 6A ) 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 . 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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Figure 6A) 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.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.


Figure 7A ) Representative images showing G1 localization of human M18BP1WT or human M18BP1SANTA in sfGFP - AID - M18BP1 DLD1 cells treated with 1 mM IAA for 24 h to remove endogenous M18BP1 . Centromeric localization is indicated by localization with ACA ( α - centromere autoantibody serum ) , early G1 cell cycle state is indicated by midbody staining in the tubulin channel . M18BP1 species indicated at left , immunolocalized protein indicated above . Scale bar , 10 μm . Insets are magnified 3X . B ) Representative immunofluorescence images showing localization of full - length Flag - M18BP1 - 1 ( mutant species indicated at left ) without ( left panels ) and with ( right panels ) fusion to an SV40 NLS to sperm centromeres in interphase extract depleted of endogenous M18BP1 . Immunolocalized protein indicated above . Scale bar , 10 μm . Insets are magnified 3X . Quantification of immunofluorescence intensity at centromeres as total integrated fluorescence signal normalized to wild type levels . The values shown on each panel are represented as a percentage of wild - type Flag - M18BP1 - 1 ± SEM from three independent experiments . C ) Quantification of Myc - CENP - A assembly in M18BP1 - depleted extract complemented with the indicated M18BP1 - 1 species . Graph shows mean immunofluorescence intensity at centromeres normalized to reactions complemented with WT M18BP1 - 1 . Error bars show SEM of at least three independent experiments . D ) Representative Western blot of CENP - A assembly reactions in ( B ) . Efficient M18BP1 depletion is indicated by comparing lanes 1 and 10 . Add - back of wild - type or mutant M18BP1 - 1 is near endogenous levels .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) Representative images showing G1 localization of human M18BP1WT or human M18BP1SANTA in sfGFP-AID-M18BP1 DLD1 cells treated with 1 mM IAA for 24 h to remove endogenous M18BP1. Centromeric localization is indicated by localization with ACA (α-centromere autoantibody serum), early G1 cell cycle state is indicated by midbody staining in the tubulin channel. M18BP1 species indicated at left, immunolocalized protein indicated above. Scale bar, 10 μm. Insets are magnified 3X.B) Representative immunofluorescence images showing localization of full-length Flag-M18BP1-1 (mutant species indicated at left) without (left panels) and with (right panels) fusion to an SV40 NLS to sperm centromeres in interphase extract depleted of endogenous M18BP1. Immunolocalized protein indicated above. Scale bar, 10 μm. Insets are magnified 3X. Quantification of immunofluorescence intensity at centromeres as total integrated fluorescence signal normalized to wild type levels. The values shown on each panel are represented as a percentage of wild-type Flag-M18BP1-1 ± SEM from three independent experiments.C) Quantification of Myc-CENP-A assembly in M18BP1-depleted extract complemented with the indicated M18BP1-1 species. Graph shows mean immunofluorescence intensity at centromeres normalized to reactions complemented with WT M18BP1-1. Error bars show SEM of at least three independent experiments.D) Representative Western blot of CENP-A assembly reactions in (B). Efficient M18BP1 depletion is indicated by comparing lanes 1 and 10. Add-back of wild-type or mutant M18BP1-1 is near endogenous levels.


figf1a , Detection of wild - type ( + ) and Ambra1 gene - trap transcripts ( gt ) from total E14 . 5 embryos analysed by northern blotting . Size marker , 28S ribosomal RNA . b , Immunoblot analysis of E14 . 5embryobrain extracts using antibodies against Ambra1 ( left panel ) and β - gal ( right panel ) . Specific bands ( arrowheads ) and unspecific signals ( asterisk ) are indicated . Molecular weights are measured in kilodaltons ( kDa ) . The fusion protein can be revealed only by means of the more sensitive anti - β - gal antibody . c - f , Expression of Ambra1 in the mouse embryonic nervous system . β - gal - staining on whole - mount Ambra1 + / gt mouseembryos at E8 . 5 ( c ) , E11 . 5 ( d ) and on a cross - section of E11 . 5spinal cord ( e ) is shown . Fb , forebrain ; Hb , hindbrain ; Mb , midbrain . f , Matching expression of the endogenous gene was revealed by whole - mount Ambra1 mRNA in situ hybridization analysis in E11 . 5 wild - type embryos . g - l , Electron scanning microscopy analysis of E11 . 5 ( g ) and E12 . 5 ( h ) Ambra1gt / gt embryos . Note the failure of the neural tube closure , the extensive midbrain / hindbrain exencephaly ( Ex ) with a closed telencephalon ( T ) , and the lumbosacral spina bifida ( Sb ) . i , j , E14 . 5 wild - type ( i ) and Ambra1gt / gt ( j ) embryos are characterized by prominent exencephaly . k , l , Histological analysis of E12 . 5 wild - type ( k ) and Ambra1gt / gt ( l ) embryos on cross - section . Note the absence of a normal ventricular system , the extensive overgrowth of the proliferative neuroepithelium in the diencephalon ( Di ) and spinal cord ( Sc ) , and the enlarged fifth ganglia ( VG ) in the Ambra1gt / gt embryo . m - p , TUNEL ( TdT - mediated dUTP nick end labelling ) staining of E10 . 5brain ( m , n ) and E9 . 5spinal cord ( o , p ) in wild - type ( m , o ) and Ambra1gt / gt ( n , p ) embryo sections . q - t , Analysis of cell proliferation in wild - type ( q , s ) and Ambra1gt / gt ( r , t ) embryos on transverse sections of E8 . 5cephalic neural folds in prospective hindbrain region ( mitoses , arrows in q , r ) and on sagittal sections of E10 . 5forebrain ( BrdU uptake , s , t ) .

B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1a, Detection of wild-type (+) and Ambra1 gene-trap transcripts (gt) from total E14.5 embryos analysed by northern blotting. Size marker, 28S ribosomal RNA.b, Immunoblot analysis of E14.5embryobrain extracts using antibodies against Ambra1 (left panel) and β-gal (right panel). Specific bands (arrowheads) and unspecific signals (asterisk) are indicated. Molecular weights are measured in kilodaltons (kDa). The fusion protein can be revealed only by means of the more sensitive anti-β-gal antibody.c-f, Expression of Ambra1 in the mouse embryonic nervous system. β-gal-staining on whole-mount Ambra1+/gt mouseembryos at E8.5 (c), E11.5 (d) and on a cross-section of E11.5spinal cord (e) is shown. Fb, forebrain; Hb, hindbrain; Mb, midbrain. f, Matching expression of the endogenous gene was revealed by whole-mount Ambra1 mRNA in situ hybridization analysis in E11.5 wild-type embryos.g-l, Electron scanning microscopy analysis of E11.5 (g) and E12.5 (h) Ambra1gt/gt embryos. Note the failure of the neural tube closure, the extensive midbrain/hindbrain exencephaly (Ex) with a closed telencephalon (T), and the lumbosacral spina bifida (Sb). i, j, E14.5 wild-type (i) and Ambra1gt/gt (j) embryos are characterized by prominent exencephaly. k, l, Histological analysis of E12.5 wild-type (k) and Ambra1gt/gt (l) embryos on cross-section. Note the absence of a normal ventricular system, the extensive overgrowth of the proliferative neuroepithelium in the diencephalon (Di) and spinal cord (Sc), and the enlarged fifth ganglia (VG) in the Ambra1gt/gt embryo.m-p, TUNEL (TdT-mediated dUTP nick end labelling) staining of E10.5brain (m, n) and E9.5spinal cord (o, p) in wild-type (m, o) and Ambra1gt/gt (n, p) embryo sections. q-t, Analysis of cell proliferation in wild-type (q, s) and Ambra1gt/gt (r, t) embryos on transverse sections of E8.5cephalic neural folds in prospective hindbrain region (mitoses, arrows in q, r) and on sagittal sections of E10.5forebrain (BrdU uptake, s, t).


figf2a , AMBRA1 - BECN1 interaction by yeast two - hybrid assay . Yeast cells were co - transfected with the indicated plasmids and plated in medium with ( + ) or without ( - ) histidine ( H ) and adenine ( A ) . AD , activation domain ; DBD , DNA - binding domain ; G4 , Gal4 ; LAM , lamin . b , AMBRA1 - BECN1 interaction in mammalian cells . 2FTGH cells were co - infected with retroviral vectors encoding BECN1 and the indicated Myc - tagged AMBRA1 proteins , or Myc - tagged β - gal as a negative control . CT , C terminal ; FL , full length ; NT , N terminal . Protein extracts were immunoprecipitated using an anti - Myc - tagged antibody ( IP Myc ) . Purified complexes and corresponding total extracts were analysed by western blot using anti - Myc ( WB Myc , upper panels ) or anti - BECN1 ( WB BECN1 , lower panels ) antibodies . When multiple bands are present , the upper band has the expected molecular mass . A scheme of the various AMBRA1 mutants is reported . c , AMBRA1 is in a complex that includes BECN1 and Vps34 . Protein extracts from 2FTGH cells co - expressing BECN1 and the Myc - tagged AMBRA1 full - length protein ( FL ) or Myc - tagged β - gal were immunoprecipitated using an anti - Myc - tag antibody ( IP Myc ) . Purified complexes and corresponding total extracts were analysed using anti - BECN1 ( WB BECN1 ) and anti - Vps34 ( WB Vps34 ) antibodies . b , Immunoblot analysis of E14 . 5embryobrain extracts using antibodies against Ambra1 ( left panel ) and β - gal ( right panel ) . Specific bands ( arrowheads ) and unspecific signals ( asterisk ) are indicated . Molecular weights are measured in kilodaltons ( kDa ) . The fusion protein can be revealed only by means of the more sensitive anti - β - gal antibody . e , BECN1 and AMBRA1 co - localize in mammalian cells . 2FTGH cells co - expressing BECN1 and Myc - tagged AMBRA1 were stained for Myc ( green ) and BECN1 ( red ) . Scale bars , 8 µm .

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figf2a, AMBRA1-BECN1 interaction by yeast two-hybrid assay. Yeast cells were co-transfected with the indicated plasmids and plated in medium with (+) or without (-) histidine (H) and adenine (A). AD, activation domain; DBD, DNA-binding domain; G4, Gal4; LAM, lamin.b, AMBRA1-BECN1 interaction in mammalian cells. 2FTGH cells were co-infected with retroviral vectors encoding BECN1 and the indicated Myc-tagged AMBRA1 proteins, or Myc-tagged β-gal as a negative control. CT, C terminal; FL, full length; NT, N terminal. Protein extracts were immunoprecipitated using an anti-Myc-tagged antibody (IP Myc). Purified complexes and corresponding total extracts were analysed by western blot using anti-Myc (WB Myc, upper panels) or anti-BECN1 (WB BECN1, lower panels) antibodies. When multiple bands are present, the upper band has the expected molecular mass. A scheme of the various AMBRA1 mutants is reported.c, AMBRA1 is in a complex that includes BECN1 and Vps34. Protein extracts from 2FTGH cells co-expressing BECN1 and the Myc-tagged AMBRA1 full-length protein (FL) or Myc-tagged β-gal were immunoprecipitated using an anti-Myc-tag antibody (IP Myc). Purified complexes and corresponding total extracts were analysed using anti-BECN1 (WB BECN1) and anti-Vps34 (WB Vps34) antibodies.b, Immunoblot analysis of E14.5embryobrain extracts using antibodies against Ambra1 (left panel) and β-gal (right panel). Specific bands (arrowheads) and unspecific signals (asterisk) are indicated. Molecular weights are measured in kilodaltons (kDa). The fusion protein can be revealed only by means of the more sensitive anti-β-gal antibody.e, BECN1 and AMBRA1 co-localize in mammalian cells. 2FTGH cells co-expressing BECN1 and Myc-tagged AMBRA1 were stained for Myc (green) and BECN1 (red). Scale bars, 8 µm.


figf3a , AMBRA1 downregulation in 2FTGH cells using specific short interfering RNA ( siRNA ) oligoribonucleotides ( 1 and 2 ) . AMBRA1 mRNA and protein levels were analysed by quantitative polymerase chain reaction ( PCR , left ) and western blotting ( right ) , respectively . An unrelated oligoribonucleotide was used as the control . Tubulin was used as the protein - loading control . RL , relative levels . The arrowhead indicates the AMBRA1 - specific band . b , Rapamycin - induced autophagy requires AMBRA1 . After AMBRA1 downregulation , 2FTGH cells were treated with rapamycin ; after 48 h , the occurrence of autophagy was analysed by appearance of GFP - LC3 punctate staining or by LC3 - I to LC3 - II conversion ( western blot in inset ) . Representative results are accompanied by a graph reporting data from three experiments . Scale bar , 20 µm . c , Nutrient - starvation - induced autophagy requires AMBRA1 . After AMBRA1 downregulation , 2FTGH cells were starved for 4 h and analysed for appearance of GFP - LC3 punctate staining . d , AMBRA1 overexpression increases basal and rapamycin - induced autophagy . 2FTGH cells were transduced with full - length AMBRA1 ( FL ) , AMBRA1 fragments ( F1 - 3 ) , BECN1 and β - gal ( negative control ) encoding retroviruses . They were then stimulated with rapamycin or left untreated , and analysed by appearance of GFP - LC3 punctate staining . e , BECN1 - induced autophagy requires AMBRA1 . After AMBRA1 downregulation , GFP - LC3 - expressing 2FTGH cells were transduced with a BECN1 - encoding retrovirus . Occurrence of autophagy was analysed 48 h later by appearance of GFP - LC3 punctate staining . f , AMBRA1 downregulation reduces the amount of Vps34 associated to BECN1 during autophagy . Forty - eight hours after AMBRA1 downregulation with oligo 2 , 2FTGH cells were either starved or cultured in standard medium for an additional 4 h . Protein extracts were immunoprecipitated ( IP ) using an anti - BECN1 antibody . Purified complexes and corresponding total extracts were analysed by western blotting with anti - Vps34 ( left panels ) and anti - BECN1 ( right panels ) antibodies . The graph indicates the signal intensity of BECN1 - associated Vps34 as determined by densitometry .

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figf3a, AMBRA1 downregulation in 2FTGH cells using specific short interfering RNA (siRNA) oligoribonucleotides (1 and 2). AMBRA1 mRNA and protein levels were analysed by quantitative polymerase chain reaction (PCR, left) and western blotting (right), respectively. An unrelated oligoribonucleotide was used as the control. Tubulin was used as the protein-loading control. RL, relative levels. The arrowhead indicates the AMBRA1-specific band.b, Rapamycin-induced autophagy requires AMBRA1. After AMBRA1 downregulation, 2FTGH cells were treated with rapamycin; after 48 h, the occurrence of autophagy was analysed by appearance of GFP-LC3 punctate staining or by LC3-I to LC3-II conversion (western blot in inset). Representative results are accompanied by a graph reporting data from three experiments. Scale bar, 20 µm.c, Nutrient-starvation-induced autophagy requires AMBRA1. After AMBRA1 downregulation, 2FTGH cells were starved for 4 h and analysed for appearance of GFP-LC3 punctate staining.d, AMBRA1 overexpression increases basal and rapamycin-induced autophagy. 2FTGH cells were transduced with full-length AMBRA1 (FL), AMBRA1 fragments (F1-3), BECN1 and β-gal (negative control) encoding retroviruses. They were then stimulated with rapamycin or left untreated, and analysed by appearance of GFP-LC3 punctate staining.e, BECN1-induced autophagy requires AMBRA1. After AMBRA1 downregulation, GFP-LC3-expressing 2FTGH cells were transduced with a BECN1-encoding retrovirus. Occurrence of autophagy was analysed 48 h later by appearance of GFP-LC3 punctate staining.f, AMBRA1 downregulation reduces the amount of Vps34 associated to BECN1 during autophagy. Forty-eight hours after AMBRA1 downregulation with oligo 2, 2FTGH cells were either starved or cultured in standard medium for an additional 4 h. Protein extracts were immunoprecipitated (IP) using an anti-BECN1 antibody. Purified complexes and corresponding total extracts were analysed by western blotting with anti-Vps34 (left panels) and anti-BECN1 (right panels) antibodies. The graph indicates the signal intensity of BECN1-associated Vps34 as determined by densitometry.


figf4a , Alteration of both LC3 conversion and translocation in Ambra1gt / gt embryos . Left panel : immunoblot analysis of LC3 in E14 . 5embryos . LC3 - I to LC3 - II conversion is reduced in mutant extracts ( LC3 - II / LC3 - I ratio , as measured by densitometry , from left to right : 5 % , 9 . 8 % , 2 . 5 % , 4 . 6 % ) . B , body ; H , head . Centre and right panels : sections from E10 . 5Ambra1 + / + ; GFP - LC3 and Ambra1gt / gt ; GFP - LC3neuroepithelia were compared to evaluate GFP - LC3 subcellular distribution . Clusters of GFP - LC3 punctate structures ( arrows ) are indicated in wild - type sections . Images were optimized by deconvolution . Scale bar , 10 µm . c , Rapamycin - induced autophagy is impaired in Ambra1gt / gt murine embryonic fibroblasts ( MEFs ) . Cells dissected from embryos with different genotypes ( A - D ) were treated with rapamycin , and the occurrence of autophagy was analysed 48 h later by the appearance of GFP - LC3 punctate staining . Representative results are shown in the upper panels , whereas the lower panel shows the data from three experiments . Scale bar , 20 µm . a , AMBRA1 - BECN1 interaction by yeast two - hybrid assay . Yeast cells were co - transfected with the indicated plasmids and plated in medium with ( + ) or without ( - ) histidine ( H ) and adenine ( A ) . AD , activation domain ; DBD , DNA - binding domain ; G4 , Gal4 ; LAM , lamin .

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figf4a, Alteration of both LC3 conversion and translocation in Ambra1gt/gt embryos. Left panel: immunoblot analysis of LC3 in E14.5embryos. LC3-I to LC3-II conversion is reduced in mutant extracts (LC3-II/LC3-I ratio, as measured by densitometry, from left to right: 5%, 9.8%, 2.5%, 4.6%). B, body; H, head. Centre and right panels: sections from E10.5Ambra1+/+;GFP-LC3 and Ambra1gt/gt;GFP-LC3neuroepithelia were compared to evaluate GFP-LC3 subcellular distribution. Clusters of GFP-LC3 punctate structures (arrows) are indicated in wild-type sections. Images were optimized by deconvolution. Scale bar, 10 µm.c, Rapamycin-induced autophagy is impaired in Ambra1gt/gt murine embryonic fibroblasts (MEFs). Cells dissected from embryos with different genotypes (A-D) were treated with rapamycin, and the occurrence of autophagy was analysed 48 h later by the appearance of GFP-LC3 punctate staining. Representative results are shown in the upper panels, whereas the lower panel shows the data from three experiments. Scale bar, 20 µm.a, AMBRA1-BECN1 interaction by yeast two-hybrid assay. Yeast cells were co-transfected with the indicated plasmids and plated in medium with (+) or without (-) histidine (H) and adenine (A). AD, activation domain; DBD, DNA-binding domain; G4, Gal4; LAM, lamin.


Figure 1D , RT - qPCR time courses . Promoters of the indicated oscillating genes were used to drive expression of a destabilized , nuclear GFP protein from a single copy integrated transgene ; gfp mRNA and the endogenous transcript driven by the same promoter were quantified from the same RNA samples . Relative expression was calculated as - dCT = - ( target CT values - actin CT values ) and then mean normalized for each trace individually . Peak phases ( ϕPeak ) and amplitudes ( A ) for the endogenous transcripts are from ( Meeuse et al . , 2020 ) . qPCR was performed in technical replicate , shown are averages .

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Figure 1D, RT-qPCR time courses. Promoters of the indicated oscillating genes were used to drive expression of a destabilized, nuclear GFP protein from a single copy integrated transgene; gfp mRNA and the endogenous transcript driven by the same promoter were quantified from the same RNA samples. Relative expression was calculated as -dCT = - (target CT values - actin CT values) and then mean normalized for each trace individually. Peak phases (ϕPeak) and amplitudes (A) for the endogenous transcripts are from (Meeuse et al., 2020). qPCR was performed in technical replicate, shown are averages.


Figure 2B , C Heatmap showing trend - corrected luminescence as in ( A ) , for indicated RNAi conditions causing altered numbers ( B ) or durations ( C ) of molts , respectively .

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Figure 2B, C Heatmap showing trend-corrected luminescence as in (A), for indicated RNAi conditions causing altered numbers (B) or durations (C) of molts, respectively.


Figure 3C , Image sequence of an L1 synchronized grh - 1 : : aid animal ( HW2418 ) grown at 20 ° C on a 250 μM auxin - containing plates . A lethargic animal was transferred to an agar - pad containing microscopy slide and images were collected every 1 sec , using DIC , 100x magnification . Selected images of Movie EV1 are shown . Time stamp ( min : sec ) is indicated . Arrows indicate phenotypic features : loosening of the cuticle ( 0 : 00 ) ; back - and - forth movements ( 0 : 05 ) ; inflation of the cuticle ( 2 : 46 ) ; vesicles underneath loosened cuticle ( 6 : 11 ) ; rupturing of the cuticle ( 6 : 20 , 6 : 40 ) .

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Figure 3C, Image sequence of an L1 synchronized grh-1::aid animal (HW2418) grown at 20°C on a 250 μM auxin-containing plates. A lethargic animal was transferred to an agar-pad containing microscopy slide and images were collected every 1 sec, using DIC, 100x magnification. Selected images of Movie EV1 are shown. Time stamp (min:sec) is indicated. Arrows indicate phenotypic features: loosening of the cuticle (0:00); back-and-forth movements (0:05); inflation of the cuticle (2:46); vesicles underneath loosened cuticle (6:11); rupturing of the cuticle (6:20, 6:40).


Figure 4A , Quantification of the percentage of grh - 1 : : aid animals constitutively expressing luciferase ( HW2434 ) that enter each of four molts molt upon hatching into increasing concentrations of auxin as indicated . B , Boxplot showing the duration of M1 , M2 and M3 of animals treated with indicated concentrations of auxin . Animals that failed to develop beyond M3 in ( A ) were excluded . The horizontal line represents the median , hinges extend to first and third quartiles , and the whiskers extend up to the most extreme value within 1 . 5 * IQR ( interquartile range ) of the median . Significant differences relative to 0 nM auxin are indicated . P - values were determined by a non - parametric Wilcoxon rank sum test that does not assume normal distribution .

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Figure 4A, Quantification of the percentage of grh-1::aid animals constitutively expressing luciferase (HW2434) that enter each of four molts molt upon hatching into increasing concentrations of auxin as indicated.B, Boxplot showing the duration of M1, M2 and M3 of animals treated with indicated concentrations of auxin. Animals that failed to develop beyond M3 in (A) were excluded. The horizontal line represents the median, hinges extend to first and third quartiles, and the whiskers extend up to the most extreme value within 1.5*IQR (interquartile range) of the median. Significant differences relative to 0 nM auxin are indicated. P-values were determined by a non-parametric Wilcoxon rank sum test that does not assume normal distribution.


Figure 5A - D , Heatmaps showing trend - corrected luminescence ( Lum , arbitrary units ) of grh - 1 : : aid animals constitutively expressing luciferase ( HW2434 ) . T = 0h corresponds to time of plating embryos , which subsequently hatch at different times . Arrow indicates time point when 250 μM auxin was added , i . e . prior to the first molt ( A ; M1 ) , M2 ( B ) , M3 ( C ) or M4 ( D ) larval stage . Note that for technical convenience in ( A ) , auxin was provided at time of plating . Animals are sorted by entry into M1 ( A ) , M2 ( B ) , M3 ( C ) , M4 ( D ) , respectively .

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Figure 5A-D, Heatmaps showing trend-corrected luminescence (Lum, arbitrary units) of grh-1::aid animals constitutively expressing luciferase (HW2434). T = 0h corresponds to time of plating embryos, which subsequently hatch at different times. Arrow indicates time point when 250 μM auxin was added, i.e. prior to the first molt (A; M1), M2 (B), M3 (C) or M4 (D) larval stage. Note that for technical convenience in (A), auxin was provided at time of plating. Animals are sorted by entry into M1 (A), M2 (B), M3 (C), M4 (D), respectively.


Figure 6Time - lapse imaging of grh - 1 : : gfp : : 3xflag animals producing endogenously GFP - tagged GRH - 1 protein . Average + / - 95 % confidence interval ( cyan shading ) are shown ; gray boxes indicate average time of molts .

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Figure 6Time-lapse imaging of grh-1::gfp::3xflag animals producing endogenously GFP-tagged GRH-1 protein. Average +/- 95% confidence interval (cyan shading) are shown; gray boxes indicate average time of molts.


Figure 7D , Western blot revealing rapid GRH - 1 depletion in the grh - 1 : : aid strain ( HW2434 ) upon addition of 250 µM auxin . A synchronized culture of animals was grown in liquid at 20 ° C . After 21 h ( denoted t = 0 h in the figure ) , the culture was split in two and either auxin or vehicle were added as indicated . Cultures were sampled hourly and protein lysates probed by Western blotting using anti - FLAG and anti - actin antibodies as indicated .

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Figure 7D, Western blot revealing rapid GRH-1 depletion in the grh-1::aid strain (HW2434) upon addition of 250 µM auxin. A synchronized culture of animals was grown in liquid at 20°C. After 21 h (denoted t=0 h in the figure), the culture was split in two and either auxin or vehicle were added as indicated. Cultures were sampled hourly and protein lysates probed by Western blotting using anti-FLAG and anti-actin antibodies as indicated.


Figure 8A , Scatter plot of GRH - 1 : : GFP : : 3xFLAG ChIP - seq peak enrichments across two biological replicates , using an anti - GFP antibody to immunoprecipitate the endogenously tagged protein from L2 stage larval lysates . Peaks assigned to genes encoding rhythmically expressed transcription factors are plotted in red , all others in blue . Peaks assigned to candidate core clock genes identified in this study are labeled .

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Figure 8A, Scatter plot of GRH-1::GFP::3xFLAG ChIP-seq peak enrichments across two biological replicates, using an anti-GFP antibody to immunoprecipitate the endogenously tagged protein from L2 stage larval lysates. Peaks assigned to genes encoding rhythmically expressed transcription factors are plotted in red, all others in blue. Peaks assigned to candidate core clock genes identified in this study are labeled.


Figure 1 ( A ) GalNAc - T expression levels in human liver ( The body map data for human liver was kindly provided by the " Gene Expression Applications research group at Illumina " , http : / / www . ebi . ac . uk / arrayexpress / experiments / E - MTAB - 513 / ) and HepG2 wild type cells quantified by RNAseq ( n = 2 , bars representing mean with SD ) . ( B ) VVA lectin immunoblot of secretomes from HepG2 SC , SCΔT1 , SCΔT2 and SCΔT3 .

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Figure 1(A) GalNAc-T expression levels in human liver (The body map data for human liver was kindly provided by the "Gene Expression Applications research group at Illumina", http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-513/) and HepG2 wild type cells quantified by RNAseq (n=2, bars representing mean with SD).(B) VVA lectin immunoblot of secretomes from HepG2 SC, SCΔT1, SCΔT2 and SCΔT3.


Figure 3Histograms showing the distribution of M / L quantitation ratios of monoglycosylated peptides identified in ( A ) HepG2SCΔT1 , ( B ) ΔT2 and ( C ) + T3 . Glycopeptides with M / L ratio < - 1 are colored red and glycopeptides with a M / L ratio > + 1 are colored green . ( D ) Venn diagram showing the distribution of candidates for isoform - specific sites among HepG2SCΔT1 , T2 and + T3 applying a log10 ( + / - 1 ) cut - off ( excluding sites identified in both TCL and SEC for each isoform ) , and ( E ) TCL alone and ( F ) SEC alone .

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Figure 3Histograms showing the distribution of M/L quantitation ratios of monoglycosylated peptides identified in (A) HepG2SCΔT1, (B) ΔT2 and (C) +T3. Glycopeptides with M/L ratio < -1 are colored red and glycopeptides with a M/L ratio > +1 are colored green. (D) Venn diagram showing the distribution of candidates for isoform-specific sites among HepG2SCΔT1, T2 and +T3 applying a log10 (+/-1) cut-off (excluding sites identified in both TCL and SEC for each isoform), and (E) TCL alone and (F) SEC alone.


Figure 1DFL inhibits HMGB1 - induced , but not fMLP - induced cell migration . Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers , 1 nM HMGB1 or no chemoattractant was added in the lower chamber , together with the indicated concentrations of DFL . Data points with average ± standard deviation ( avg ± sd ; n = 3 , each point represents a biological replicate ) in a representative experiment . Histogram showing residue - specific CSPs of 15N - labelled HMGB1 ( ~ 0 . 1 mM ) upon addition of ten - fold excess of DFL ( helices are schematically represented on the top ) . Missing residues are prolines or are absent because of exchange with the solvent . Box A and Box B residues with CSP > avg + sd are represented in magenta and blue , respectively . The R23A / R109A HMGB1 double mutant has chemotactic activity but is not inhibited by DFL ( the experiment was carried out as described in panel B ) . The data points represent three biological replicates in one experiment ( of 2 performed in different days ) ; the avg ± sd is shown .

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Figure 1DFL inhibits HMGB1-induced, but not fMLP-induced cell migration. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers, 1 nM HMGB1 or no chemoattractant was added in the lower chamber, together with the indicated concentrations of DFL. Data points with average ± standard deviation (avg ± sd; n=3, each point represents a biological replicate) in a representative experiment.Histogram showing residue-specific CSPs of 15N-labelled HMGB1 (~0.1 mM) upon addition of ten-fold excess of DFL (helices are schematically represented on the top). Missing residues are prolines or are absent because of exchange with the solvent. Box A and Box B residues with CSP > avg + sd are represented in magenta and blue, respectively.The R23A/R109A HMGB1 double mutant has chemotactic activity but is not inhibited by DFL (the experiment was carried out as described in panel B). The data points represent three biological replicates in one experiment (of 2 performed in different days); the avg ± sd is shown.


Figure 2Histogram showing the CSPs of 15N - labelled CXCL12 amides ( ~ 0 . 1 mM ) upon addition of ten - fold excess of DFL . Missing residues are prolines . Elements of secondary structure are depicted on top . Top : 1H spectrum of DFL , where the numbered peaks correspond to DFL protons ( left ) . Middle : superimposition of STD spectra obtained for 0 . 5 mM DFL with 0 . 05 mM CXCL12 ( black line ) and upon addition of 0 . 2 mM of CXCR41 - 38sY21 ( red line ) . Bottom : waterLOGSY spectra obtained for 0 . 5 mM DFL with 0 . 05 mM CXCL12 ( black line ) upon addition of 0 . 2 mM of CXCR41 - 38sY21 ( red line ) . DFL does not inhibit CXCL12 induced chemotaxis . Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers ; 1 nM CXCL12 or no chemoattractant was added in the lower chamber , together with the indicated concentrations of DFL . Data points ( n = 3 ) with avg ± sd in one representative experiment ( of 2 performed in different days ) ; the migration in the presence of increasing concentrations of DFLDFL inhibits chemotaxis toward the HMGB1 / CXCL12 heterocomplex . Data points ( n = 3 ) with avg ± sd in one representative experiment ( of 3 performed in different days ) . Migration in the presence or absence of DFL is significantly different ( p = 0 . 0037 , one - way ANOVA plus Dunnett ' s post test ; * * p < 0 . 01 relative to no DFL addition ) .

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Figure 2Histogram showing the CSPs of 15N-labelled CXCL12 amides (~0.1 mM) upon addition of ten-fold excess of DFL. Missing residues are prolines. Elements of secondary structure are depicted on top.Top: 1H spectrum of DFL, where the numbered peaks correspond to DFL protons (left). Middle: superimposition of STD spectra obtained for 0.5 mM DFL with 0.05 mM CXCL12 (black line) and upon addition of 0.2 mM of CXCR41-38sY21 (red line). Bottom: waterLOGSY spectra obtained for 0.5 mM DFL with 0.05 mM CXCL12 (black line) upon addition of 0.2 mM of CXCR41-38sY21 (red line).DFL does not inhibit CXCL12 induced chemotaxis. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers; 1 nM CXCL12 or no chemoattractant was added in the lower chamber, together with the indicated concentrations of DFL. Data points (n=3) with avg ± sd in one representative experiment (of 2 performed in different days); the migration in the presence of increasing concentrations of DFLDFL inhibits chemotaxis toward the HMGB1/CXCL12 heterocomplex. Data points (n=3) with avg ± sd in one representative experiment (of 3 performed in different days). Migration in the presence or absence of DFL is significantly different (p=0.0037, one-way ANOVA plus Dunnett's post test; ** p<0.01 relative to no DFL addition).


Figure 31H - 15N HSQC HMGB1 ( 0 . 1 mM ) spectrum1H - 15N HSQC HMGB1 ( 0 . 1 mM ) spectrum with 0 . 2 mM CXCL12 . MST measurements of CXCL12 titrated into 50 nM fluorescently labelled HMGB1 ; the signal increases from ∼ 930 ( free ) to ∼ 950 a . u . ( bound ) yielding an apparent Kd of 4 ± 0 . 4 μM . n = 3 ; error bars represent avg ± sd . H - 15N HSQC of HMGB1 ( 0 . 1 mM ) with CXCL12 ( 0 . 2 mM ) upon addition of ( D ) 0 . 2 mM and ( E ) 1 mM DFL . MST measurements of the heterocomplex ( preformed using 50 nM HMGB1 and 50 μM CXCL12 ) in the presence of increasing concentrations of DFL . Decreasing MST signal ( from ∼ 950 to ∼ 930 a . u . ) upon DFL titration indicates that CXCL12 has been displaced from HMGB1 . n = 3 ; error bars represent avg ± sd .

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Figure 31H-15N HSQC HMGB1 (0.1 mM) spectrum1H-15N HSQC HMGB1 (0.1 mM) spectrum with 0.2 mM CXCL12.MST measurements of CXCL12 titrated into 50 nM fluorescently labelled HMGB1; the signal increases from ∼930 (free) to ∼950 a.u. (bound) yielding an apparent Kd of 4 ± 0.4 μM. n=3; error bars represent avg ± sd.H-15N HSQC of HMGB1 (0.1 mM) with CXCL12 (0.2 mM) upon addition of (D) 0.2 mM and (E) 1 mM DFL.MST measurements of the heterocomplex (preformed using 50 nM HMGB1 and 50 μM CXCL12) in the presence of increasing concentrations of DFL. Decreasing MST signal (from ∼950 to ∼930 a.u.) upon DFL titration indicates that CXCL12 has been displaced from HMGB1. n=3; error bars represent avg ± sd.


Figure 4DFL does not affect the cytokine - inducing activities of disulfide HMGB1 ( ds - HMGB1 ) . Mouse Bone Marrow Derived Macrophages were activated or not for 3 h with 3 µg / ml ds - HMGB1 ( ~ 100 nM ) or 10 ng / ml LPS , in the presence of the indicated concentrations of DFL . The data points represent n = 3 biological replicates , with avg ± sd , in one representative experiment ( of 2 performed in different days ) . The levels of TNFα mRNA are not significantly different in the presence of increasing concentrations of DFL ( one - way ANOVA plus post tests ) . DFL does not inhibit chemotaxis towards 3S . Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers ; 1 nM fully reduced HMGB1 ( fr - HMGB1 ) ( green ) or 3S ( red ) , or no chemoattractant ( black ) , were added in the lower chamber , together with 1 µg / ml of anti - CXCL12 monoclonal antibody or 30 nM DFL . Data points ( n = 4 ) with avg ± sd in one representative experiment ( of two performed in different days ) . Statistics : one - way ANOVA ( p < 0 . 0001 ) , followed by Dunnett ' s post - test . * * * , p < 0 . 001 relative to migration towards HMGB1 , the migrations towards 3S are not significantly different among each other . Quantification of CD45 + cells isolated with immunobeads from injured muscles 6 hours after damage . Before damage mice were treated either with Phosphate Buffered Saline vehicle , Glycyrrhizin or DFL . Two independent experiments . Each point represents a different mouse , avg ± sd are shown ( statistics : Kruskal - Willis plus post - tests ) .

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Figure 4DFL does not affect the cytokine-inducing activities of disulfide HMGB1 (ds-HMGB1). Mouse Bone Marrow Derived Macrophages were activated or not for 3 h with 3 µg/ml ds-HMGB1 (~100 nM) or 10 ng/ml LPS, in the presence of the indicated concentrations of DFL. The data points represent n=3 biological replicates, with avg ± sd, in one representative experiment (of 2 performed in different days). The levels of TNFα mRNA are not significantly different in the presence of increasing concentrations of DFL (one-way ANOVA plus post tests).DFL does not inhibit chemotaxis towards 3S. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers; 1 nM fully reduced HMGB1 (fr-HMGB1) (green) or 3S (red), or no chemoattractant (black), were added in the lower chamber, together with 1 µg/ml of anti-CXCL12 monoclonal antibody or 30 nM DFL. Data points (n=4) with avg ± sd in one representative experiment (of two performed in different days). Statistics: one-way ANOVA (p<0.0001), followed by Dunnett's post-test. ***, p<0.001 relative to migration towards HMGB1, the migrations towards 3S are not significantly different among each other.Quantification of CD45+ cells isolated with immunobeads from injured muscles 6 hours after damage. Before damage mice were treated either with Phosphate Buffered Saline vehicle, Glycyrrhizin or DFL. Two independent experiments. Each point represents a different mouse, avg ± sd are shown (statistics: Kruskal-Willis plus post-tests).


Figure 1 ( b ) Upper panel : Representative images of BOEC colonies from PBMC cultivation 10 days post - seeding . Dotted line in each image outlines a BOEC colony . Lower panel : Stabilized BOEC cultures after passaging of colonies ( scale bar , 250 µm ) . ( c ) Proliferation doubling time for NAFLD and healthy BOECs based on passages 2 and 3 . Sample sizes are n = 9 healthy , n = 7 NAFLD ; with 3 biological replicates per donor . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , not significant ( t - test ) . ( d ) Left panel : Flow cytometry characterization of BOECs for endothelial , leukocyte and progenitor cell markers ( grey - isotype control ; red / blue / green / purple - cell lineage marker staining ) . HUVEC and monocytic THP - 1 cells were used as positive controls for endothelial and leukocytic markers respectively . Right panel : Percentages of positively expressing cells . Sample sizes are n = 4 - 6 healthy , n = 2 - 7 NAFLD . ( e ) Representative images of immunostaining for endothelial markers , PECAM1 and CDH5 ( scale bar , 100 µm ) . ( f ) Left panel : Longitudinal monitoring of BOEC angiogenesis in fibrin gel bead - based sprouting assays over 24h ( representative images ; scale bar , 100 µm ) . Right panel : Box - whisker plots of quantifications of sprout lengths and numbers of sprout per bead at 24h . n = 3 healthy , n = 3 NAFLD , with 4 - 5 beads analyzed per donor and 5 - 11 sprouts per bead .

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Figure 1(b) Upper panel: Representative images of BOEC colonies from PBMC cultivation 10 days post-seeding. Dotted line in each image outlines a BOEC colony. Lower panel: Stabilized BOEC cultures after passaging of colonies (scale bar, 250 µm). (c) Proliferation doubling time for NAFLD and healthy BOECs based on passages 2 and 3. Sample sizes are n = 9 healthy, n = 7 NAFLD; with 3 biological replicates per donor. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, not significant (t-test).(d) Left panel: Flow cytometry characterization of BOECs for endothelial, leukocyte and progenitor cell markers (grey - isotype control; red/blue/green/purple - cell lineage marker staining). HUVEC and monocytic THP-1 cells were used as positive controls for endothelial and leukocytic markers respectively. Right panel: Percentages of positively expressing cells. Sample sizes are n = 4-6 healthy, n = 2-7 NAFLD.(e) Representative images of immunostaining for endothelial markers, PECAM1 and CDH5 (scale bar, 100 µm).(f) Left panel: Longitudinal monitoring of BOEC angiogenesis in fibrin gel bead-based sprouting assays over 24h (representative images; scale bar, 100 µm). Right panel: Box-whisker plots of quantifications of sprout lengths and numbers of sprout per bead at 24h. n = 3 healthy, n = 3 NAFLD, with 4-5 beads analyzed per donor and 5-11 sprouts per bead.


Figure 2 ( b ) Volcano plot depicting differentially expressed genes comparing NAFLD BOECs with healthy BOECs . Red and blue dots represent upregulated and downregulated genes in NAFLD BOECs , respectively . ( e ) Heatmap featuring the genes in MCODE Cluster 1 .

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Figure 2(b) Volcano plot depicting differentially expressed genes comparing NAFLD BOECs with healthy BOECs. Red and blue dots represent upregulated and downregulated genes in NAFLD BOECs, respectively.(e) Heatmap featuring the genes in MCODE Cluster 1.


Figure 3 ( a ) Fold change in gene expressions of CXCL10 , CXCL11 , CXCL12 , CCL20 and CX3CL1 in NAFLD ( n = 10 ) BOECs , normalized to healthy ( n = 12 ) BOECs , with 2 technical replicates per donor . ( c ) Fold change in gene expressions of CXCL10 , CXCL11 , CXCL12 , CCL20 and CX3CL1 in LPS + FFA - treated or non - treated NAFLD ( n = 5 ) and healthy ( n = 5 ) BOECs , normalized to non - treated healthy BOECs , with 3 technical replicates per donor . ( b ) Left : Representative images showing non - treated and LPS + FFA - treated NAFLD and healthy BOECs , stained with Nile Red ( scale bars , 100 µm ) . Right : Fluorescence intensity of intracellular Nile Red stain was quantified at 515 / 585nm , and readings were normalized with blanks ( n = 3 donors / group ) . ( d ) Protein concentrations of chemokines in the conditioned culture media of healthy and NAFLD BOECs . * ( e ) Left : Representative images ( Bright field ; DAPI , nuclei ; CDH5 , endothelial cells ; CXCL12 ) of liver sections from chow diet - fed and HFHC diet - fed humanized mice ( scale bar , 40 μm ) . Boxed regions were further magnified ( scale bar , 30 µm ) . Right : Box - whisker plot of Mander ' s Coefficient ( M2 ) that was a measure of the degree of CXCL12 overlap with CDH5 - positive vasculatures in the livers . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . Chow diet - fed ( n = 4 ) and HFHC diet - fed ( n = 4 ) humanized mice were analysed , with 10 - 20 independent regions of interest per mouse were analysed for image quantification . ( f ) Left : Representative images ( Bright field ; DAPI , nuclei ; CDH5 , endothelial cells ; CXCL12 ) of aortic sections from chow diet - fed and LIDPAD mice ( scale bar , 40 μm ) . Boxed regions were further magnified ( scale bar , 30 µm ) . Arrows point at aortic endothelial cells . AW : Aortic wall ; L : lumen . Right : Box - whisker plot of CXCL12 fluorescent intensity in the aortic vascular endothelia . CXCL12 fluorescent intensity for each delimitated endothelial region is represented as individual data points on the plot . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . Chow diet - fed ( n = 3 ) and LIDPAD diet - fed ( n = 3 ) mice were analysed , with 10 - 20 delimitated endothelial regions per mouse analysed for image quantification . ( g ) Left : Representative images showing aortic sections harvested from mice fed with either chow or LIDPAD diet . Sections were stained for leukocyte markers CD45 ( red ) to determine the extent of leukocyte infiltration . Dotted lines demarcate the aortic walls ( AW ) . L : lumen . Scale bar : 20 µm . Right : Box - whisker plot of mean fluorescence intensity ( MFI ) of CD45 signals as a readout for the amount of infiltrated CD45 + leukocytes within the aortic walls . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . n = 3 mice per group , with 2 tissue sections analyzed per animal .

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Figure 3(a) Fold change in gene expressions of CXCL10, CXCL11, CXCL12, CCL20 and CX3CL1 in NAFLD (n = 10) BOECs, normalized to healthy (n = 12) BOECs, with 2 technical replicates per donor. (c) Fold change in gene expressions of CXCL10, CXCL11, CXCL12, CCL20 and CX3CL1 in LPS+FFA-treated or non-treated NAFLD (n = 5) and healthy (n = 5) BOECs, normalized to non-treated healthy BOECs, with 3 technical replicates per donor.(b) Left: Representative images showing non-treated and LPS+FFA-treated NAFLD and healthy BOECs, stained with Nile Red (scale bars, 100 µm). Right: Fluorescence intensity of intracellular Nile Red stain was quantified at 515/585nm, and readings were normalized with blanks (n = 3 donors/ group).(d) Protein concentrations of chemokines in the conditioned culture media of healthy and NAFLD BOECs. *(e) Left: Representative images (Bright field; DAPI, nuclei; CDH5, endothelial cells; CXCL12) of liver sections from chow diet-fed and HFHC diet-fed humanized mice (scale bar, 40 μm). Boxed regions were further magnified (scale bar, 30 µm). Right: Box-whisker plot of Mander's Coefficient (M2) that was a measure of the degree of CXCL12 overlap with CDH5-positive vasculatures in the livers. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). Chow diet-fed (n = 4) and HFHC diet-fed (n = 4) humanized mice were analysed, with 10-20 independent regions of interest per mouse were analysed for image quantification.(f) Left: Representative images (Bright field; DAPI, nuclei; CDH5, endothelial cells; CXCL12) of aortic sections from chow diet-fed and LIDPAD mice (scale bar, 40 μm). Boxed regions were further magnified (scale bar, 30 µm). Arrows point at aortic endothelial cells. AW: Aortic wall; L: lumen. Right: Box-whisker plot of CXCL12 fluorescent intensity in the aortic vascular endothelia. CXCL12 fluorescent intensity for each delimitated endothelial region is represented as individual data points on the plot. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). Chow diet-fed (n = 3) and LIDPAD diet-fed (n = 3) mice were analysed, with 10-20 delimitated endothelial regions per mouse analysed for image quantification.(g) Left: Representative images showing aortic sections harvested from mice fed with either chow or LIDPAD diet. Sections were stained for leukocyte markers CD45 (red) to determine the extent of leukocyte infiltration. Dotted lines demarcate the aortic walls (AW). L: lumen. Scale bar: 20 µm. Right: Box-whisker plot of mean fluorescence intensity (MFI) of CD45 signals as a readout for the amount of infiltrated CD45+ leukocytes within the aortic walls. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). n = 3 mice per group, with 2 tissue sections analyzed per animal.