Macrophages were treated for 24 h with CL097 , ssRNA40 , ssRNA41 or rapamycin ( Rapa ) . ( A ) Immunoblot of co - immunoprecipitation of cell lysates with BECN1 specific antibody using antibody to PIK3C3 and BECN1 with antibody to β - actin as loading control .

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Macrophages were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Immunoblot of co-immunoprecipitation of cell lysates with BECN1 specific antibody using antibody to PIK3C3 and BECN1 with antibody to β-actin as loading control.


( B ) Cells were incubated with 10 µg / mL pepstatin A for 4 h prior to lysis . Immunoblots of LC3B isoforms using antibody to LC3B or β - actin .

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(B) Cells were incubated with 10 µg/mL pepstatin A for 4 h prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.


( C ) Cells were fixed and permeabilized then stained with antibody to LC3B ( green ) and Hoechst 33342 ( blue ) . Scale bars indicate 10 µm . Left , representative fluorescence microscopy images . When puncta are not present , LC3B staining gives rise to a diffuse cytoplasmic pattern . Right , percentage of cells with LC3B puncta . Bar charts are presented as the means ± s . e . m . , n = 6 .

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(C) Cells were fixed and permeabilized then stained with antibody to LC3B (green) and Hoechst 33342 (blue). Scale bars indicate 10 µm. Left, representative fluorescence microscopy images. When puncta are not present, LC3B staining gives rise to a diffuse cytoplasmic pattern. Right, percentage of cells with LC3B puncta. Bar charts are presented as the means ± s.e.m., n = 6.


( D ) Immunoblots of SQSTM1 using antibody to SQSTM1 or β - actin . * p < 0 . 05 .

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(D) Immunoblots of SQSTM1 using antibody to SQSTM1 or β-actin. * p<0.05.


Macrophages transduced with non - specific scrambled shRNA ( shNS ) or TLR8 shRNA ( shTLR8 ) were treated for 24 h with CL097 , ssRNA40 , ssRNA41 or rapamycin ( Rapa ) . ( A ) Cells were lysed and immunoblot performed with antibodies to TLR8 and β - actin .

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Macrophages transduced with non-specific scrambled shRNA (shNS) or TLR8 shRNA (shTLR8) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Cells were lysed and immunoblot performed with antibodies to TLR8 and β-actin.


( B ) Immunoblots of LC3B isoforms using antibody to LC3B or β - actin .

(B) Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.


( C ) Cells were fixed and permeabilized then stained with antibody to LC3B ( green ) and Hoechst 33342 ( blue ) . Scale bars indicate 10 µm . Left , representative fluorescence microscopy images from three donors are shown . When puncta are not present , LC3B staining gives rise to a diffuse cytoplasmic pattern . Right , percentage of cells with LC3B puncta . Bar charts are presented as the means ± s . e . m . , n = 4 . * p < 0 . 05 .

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(C) Cells were fixed and permeabilized then stained with antibody to LC3B (green) and Hoechst 33342 (blue). Scale bars indicate 10 µm. Left, representative fluorescence microscopy images from three donors are shown. When puncta are not present, LC3B staining gives rise to a diffuse cytoplasmic pattern. Right, percentage of cells with LC3B puncta. Bar charts are presented as the means ± s.e.m., n = 4. * p<0.05.


A - B ) Macrophages were treated for 24 h with CL097 , ssRNA40 or ssRNA41 . ( A ) Top , qRT - PCR for CYP27B1 after 6 h . Bottom , immunoblots of CYP27B1 using antibody to CYP27B1 or β - actin .

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A-B) Macrophages were treated for 24 h with CL097, ssRNA40 or ssRNA41. (A) Top, qRT-PCR for CYP27B1 after 6 h. Bottom, immunoblots of CYP27B1 using antibody to CYP27B1 or β-actin.


( B ) Top , qRT - PCR for VDR after 6 h . Bottom , immunoblots of VDR using antibody to VDR or β - actin .

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(B) Top, qRT-PCR for VDR after 6 h. Bottom, immunoblots of VDR using antibody to VDR or β-actin.


( C - D ) Macrophages transduced with non - specific scrambled shRNA ( shNS ) , CYP27B1 shRNA ( shCYP27B1 ) or VDR shRNA ( shVDR ) were treated for 24 h with CL097 , ssRNA40 , ssRNA41 or rapamycin ( Rapa ) . ( C ) Left , immunoblots performed with antibodies to CYP27B1 , VDR and β - actin . Right , immunoblots of LC3B isoforms using antibody to LC3B or β - actin .

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(C-D) Macrophages transduced with non-specific scrambled shRNA (shNS), CYP27B1 shRNA (shCYP27B1) or VDR shRNA (shVDR) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (C) Left, immunoblots performed with antibodies to CYP27B1, VDR and β-actin. Right, immunoblots of LC3B isoforms using antibody to LC3B or β-actin.


( D ) Cells were fixed and permeabilized then stained with antibody to LC3B . Bar charts represent the percentage of cells with LC3B puncta and are presented as the means ± s . e . m . , n = 4 .

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(D) Cells were fixed and permeabilized then stained with antibody to LC3B. Bar charts represent the percentage of cells with LC3B puncta and are presented as the means ± s.e.m., n = 4.


( E ) Macrophages were treated for 24 h with CL097 , ssRNA40 , and rapamycin in the presence of 45 nmol / L or 100 nmol / L 25D3 and lysed . Immunoblots of LC3B isoforms and SQSTM1 using antibody to LC3B , SQSTM1 or β - actin . Bar charts are reported as mean ± s . e . m . , n = 4 . * p < 0 . 05 .

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(E) Macrophages were treated for 24 h with CL097, ssRNA40, and rapamycin in the presence of 45 nmol/L or 100 nmol/L 25D3 and lysed. Immunoblots of LC3B isoforms and SQSTM1 using antibody to LC3B, SQSTM1 or β-actin. Bar charts are reported as mean ± s.e.m., n = 4. * p<0.05.


Macrophages transduced with non - specific scrambled shRNA ( shNS ) or CEBPB shRNA ( shCEBPB ) were treated for 24 h with CL097 , ssRNA40 , or ssRNA41 . ( A ) Cells were lysed and immunoblot performed with antibodies to CEBPB and β - actin .

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Macrophages transduced with non-specific scrambled shRNA (shNS) or CEBPB shRNA (shCEBPB) were treated for 24 h with CL097, ssRNA40, or ssRNA41. (A) Cells were lysed and immunoblot performed with antibodies to CEBPB and β-actin.


B ) Immunoblots of CYP27B1 expression using antibody to CYP27B1 or β - actin .

B) Immunoblots of CYP27B1 expression using antibody to CYP27B1 or β-actin.


TLR8 agonists inhibit HIV replication . Macrophages were incubated with ( A ) ssRNA40 or ( B ) CL097 for 24 h before infection with HIV for 3 h . Cells were then washed and incubated with ssRNA40 or CL097 for 10 d . Extracellular release of HIV p24 antigen into the cell supernatant at days 0 , 3 , 5 , 7 , 10 was detected by ELISA . Results are reported as mean ± s . e . m . , n = 5 . * p < 0 . 05 .

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TLR8 agonists inhibit HIV replication.Macrophages were incubated with (A) ssRNA40 or (B) CL097 for 24 h before infection with HIV for 3 h. Cells were then washed and incubated with ssRNA40 or CL097 for 10 d. Extracellular release of HIV p24 antigen into the cell supernatant at days 0, 3, 5, 7, 10 was detected by ELISA. Results are reported as mean ± s.e.m., n = 5. * p<0.05.


( A ) Macrophages transduced with non - specific scrambled shRNA ( shNS ) , TLR8 shRNA ( shTLR8 ) , CYP27B1 shRNA ( shCYP27B1 ) , or VDR shRNA ( shVDR ) were treated with CL097 for 24 h before infection with HIV with continuous CL097 treatment . Top , immunoblot analysis performed using antibodies raised to TLR8 , CYP27B1 , VDR and β - actin . Bottom , ELISA performed for extracellular release of HIV p24 antigen over 10 d . Results are reported as mean ± s . e . m . , n = 4 .

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(A) Macrophages transduced with non-specific scrambled shRNA (shNS), TLR8 shRNA (shTLR8), CYP27B1 shRNA (shCYP27B1), or VDR shRNA (shVDR) were treated with CL097 for 24 h before infection with HIV with continuous CL097 treatment. Top, immunoblot analysis performed using antibodies raised to TLR8, CYP27B1, VDR and β-actin. Bottom, ELISA performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 4.


( B ) Macrophages were incubated with ssRNA40 or CL097 in the presence of 45 or 100 nmol / L 25D3 for 24 h before infection with HIV . Cells were then washed and incubated with 25D3 and ssRNA40 or CL097 for 10 d Extracellular release of HIV p24 antigen was detected by ELISA . Results are reported as mean ± s . e . m . , n = 6 . * p < 0 . 05 .

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(B) Macrophages were incubated with ssRNA40 or CL097 in the presence of 45 or 100 nmol/L 25D3 for 24 h before infection with HIV. Cells were then washed and incubated with 25D3 and ssRNA40 or CL097 for 10 d Extracellular release of HIV p24 antigen was detected by ELISA. Results are reported as mean ± s.e.m., n = 6. * p<0.05.


TLR8 - mediated inhibition of HIV is autophagy dependent . ( A - B ) Macrophages transduced with BECN1 shRNA ( shBECN1 ) ( A ) or ATG5 shRNA ( shATG5 )

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TLR8-mediated inhibition of HIV is autophagy dependent.(A-B) Macrophages transduced with BECN1 shRNA (shBECN1) (A) or ATG5 shRNA (shATG5)


( B ) were incubated with CL097 for 24 h before infection with HIV with continuous CL097 treatment for 10 d . Right , immunoblot analysis performed using antibodies raised to BECN1 , ATG5 and β - actin . Left , ELISA performed for extracellular release of HIV p24 antigen over 10 d . Results are reported as mean ± s . e . m . , n = 4 .

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(B) were incubated with CL097 for 24 h before infection with HIV with continuous CL097 treatment for 10 d. Right, immunoblot analysis performed using antibodies raised to BECN1, ATG5 and β-actin. Left, ELISA performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 4.


( C ) Macrophages were pretreated with bafilomycin A1 ( Baf A1 ) , SID 26681509 ( SID ) or vehicle control before incubation with CL097 or ssRNA40 for 24 h before infection with HIV . Cells were then washed and incubated with TLR8 agonists and inhibitors for 10 d and ELISA performed for extracellular HIV p24 antigen release over 10 d . Results are reported as mean ± s . e . m . , n = 5 . * p < 0 . 05 ; # p < 0 . 001 .

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(C) Macrophages were pretreated with bafilomycin A1 (Baf A1), SID 26681509 (SID) or vehicle control before incubation with CL097 or ssRNA40 for 24 h before infection with HIV. Cells were then washed and incubated with TLR8 agonists and inhibitors for 10 d and ELISA performed for extracellular HIV p24 antigen release over 10 d. Results are reported as mean ± s.e.m., n = 5. * p<0.05; # p<0.001.


( A ) Macrophages were treated with CL097 , ssRNA40 , ssRNA41 for 6 h after which qRT - PCR for CAMP was performed . Results are reported as mean ± s . e . m . , n = 4 .

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(A) Macrophages were treated with CL097, ssRNA40, ssRNA41 for 6 h after which qRT-PCR for CAMP was performed. Results are reported as mean ± s.e.m., n = 4.


( B ) Macrophages were treated for 24 h with CL097 or ssRNA40 in the presence of 45 nmol / L or 100 nmol / L 25D3 . Left , qRT - PCR for CAMP performed after 6 h . Results are reported as mean ± s . e . m . , n = 4 . Right , after 24 h cells were stained with anti - CAMP antibodies and analyzed by flow cytometry . Representative histograms from three donors are shown .

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(B) Macrophages were treated for 24 h with CL097 or ssRNA40 in the presence of 45 nmol/L or 100 nmol/L 25D3. Left, qRT-PCR for CAMP performed after 6 h. Results are reported as mean ± s.e.m., n = 4. Right, after 24 h cells were stained with anti-CAMP antibodies and analyzed by flow cytometry. Representative histograms from three donors are shown.


( C ) Macrophages transduced with non - specific scrambled shRNA ( shNS ) or CAMP shRNA ( shCAMP ) were incubated with CL097 , rapamycin ( Rapa ) or vehicle control for 24 h . Top , immunoblot analysis performed using antibodies raised to CAMP and β - actin . Bottom , immunoblots of LC3B isoforms using antibody to LC3B or β - actin .

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(C) Macrophages transduced with non-specific scrambled shRNA (shNS) or CAMP shRNA (shCAMP) were incubated with CL097, rapamycin (Rapa) or vehicle control for 24 h. Top, immunoblot analysis performed using antibodies raised to CAMP and β-actin. Bottom, immunoblots of LC3B isoforms using antibody to LC3B or β-actin.


( D ) After 24 h treatment with CL097 or ssRNA40 , macrophages from part C were also washed and infected with HIV and incubated with CL097 , ssRNA40 or vehicle control for 10 d . ELISA was performed for extracellular release of HIV p24 antigen over 10 d . Results are reported as mean ± s . e . m . , n = 6 . * p < 0 . 05 .

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(D) After 24 h treatment with CL097 or ssRNA40, macrophages from part C were also washed and infected with HIV and incubated with CL097, ssRNA40 or vehicle control for 10 d. ELISA was performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 6. * p<0.05.


Fate of Purkinje cells in mice with Purkinje - cell - specific knockout of Ccp1 . Ccp1flox / flox L7 - cre mice show a massive but incomplete degeneration of Purkinje cells ( calbindin - stained ; red ) at 4 months , while all Purkinje cells are degenerated at 18 months . Scale bar : 500 µm .

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Fate of Purkinje cells in mice with Purkinje-cell-specific knockout of Ccp1. Ccp1flox/flox L7-cre mice show a massive but incomplete degeneration of Purkinje cells (calbindin-stained; red) at 4 months, while all Purkinje cells are degenerated at 18 months. Scale bar: 500 µm.


Fate of Purkinje cells in mice with combinatorial knockout alleles . Knockout of Ccp1 ( pcd - mouse ; Ccp1 - / - ) results in a complete degeneration of Purkinje cells ( calbindin - stained ; red ) , while the additional knockout of Ttll1 selectively in the Purkinje cells ( L7 - cre ) protects the entire Purkinje cell layer from degeneration up to 18 months . Scale bar : 500 µm .

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Fate of Purkinje cells in mice with combinatorial knockout alleles. Knockout of Ccp1 (pcd-mouse; Ccp1-/-) results in a complete degeneration of Purkinje cells (calbindin-stained; red), while the additional knockout of Ttll1 selectively in the Purkinje cells (L7-cre) protects the entire Purkinje cell layer from degeneration up to 18 months. Scale bar: 500 µm.


Nissl staining of the Purkinje cell layer of the brains from various knockout mice shown in ( C ) confirms the absence of Purkinje cells in Ccp1 - / - , and the presence of a wild - type - like Purkinje - cell density in Ccp1 - / - Ttll1flox / flox L7 - cre mice . Scale bar : 20 µm . Purkinje cell layer ( PC ) and granule cell layer ( GC ) are indicated .

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Nissl staining of the Purkinje cell layer of the brains from various knockout mice shown in (C) confirms the absence of Purkinje cells in Ccp1-/-, and the presence of a wild-type-like Purkinje-cell density in Ccp1-/- Ttll1flox/flox L7-cre mice. Scale bar: 20 µm. Purkinje cell layer (PC) and granule cell layer (GC) are indicated.


Immunoblot of tissue extracts from cerebella of Ttll1 - / - and wild type mice , probed with the polyE , anti - ∆ 2 - tubulin and 12G10 antibodies . Increasing amounts of extracts were loaded for detection of polyglutamylated proteins with polyE .

Immunoblot of tissue extracts from cerebella of Ttll1-/- and wild type mice, probed with the polyE, anti-∆2-tubulin and 12G10 antibodies. Increasing amounts of extracts were loaded for detection of polyglutamylated proteins with polyE.


Analyses of tubulin polyglutamylation ( antibody : polyE ) in extracts of different brain regions of wild type , Ccp1 - / - , Ccp6 - / - and Ccp1 - / - Ccp6 - / - mice with immunoblot . In 3 - weeks old mice , hyperglutamylation is observed in cerebral cortex , cerebellum and hippocampus of Ccp1 - / - and Ccp1 - / - Ccp6 - / - mice . In 2 - and 5 - month - old mice , hyperglutamylation is restricted to the cerebellum in Ccp1 - / - mice , however it persists in all three brain regions of the Ccp1 - / - Ccp6 - / - mice ( complete analysis in Fig . EV1 ; α - and β - tubulin bands indicated ) .

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Analyses of tubulin polyglutamylation (antibody: polyE) in extracts of different brain regions of wild type, Ccp1-/-, Ccp6-/- and Ccp1-/-Ccp6-/- mice with immunoblot. In 3-weeks old mice, hyperglutamylation is observed in cerebral cortex, cerebellum and hippocampus of Ccp1-/- and Ccp1-/-Ccp6-/- mice. In 2- and 5-month-old mice, hyperglutamylation is restricted to the cerebellum in Ccp1-/- mice, however it persists in all three brain regions of the Ccp1-/-Ccp6-/- mice (complete analysis in Fig. EV1; α- and β-tubulin bands indicated).


Nissl - stained frontal sections of wild type , Ccp1 - / - , Ccp6 - / - and Ccp1 - / - Ccp6 - / - mice at 1 and 5 months , showing age - dependent thinning ( arrow heads ) of the cortex in Ccp1 - / - Ccp6 - / - mice ( complete analysis in Appendix Fig . S2A ) .

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Nissl-stained frontal sections of wild type, Ccp1-/-, Ccp6-/- and Ccp1-/-Ccp6-/- mice at 1 and 5 months, showing age-dependent thinning (arrow heads) of the cortex in Ccp1-/-Ccp6-/- mice (complete analysis in Appendix Fig. S2A).


Quantification of the thickness of motor and somatosensory cortex of 1 - and 5 - month - old mice shown in Appendix Fig . S2 . The single values shown in Appendix Fig . S2B were averaged , and values from each genotype - specific group were tested against the average values of the remaining three genotypes ( Appendix Fig . S2C ) . Only the cortical thickness of 5 - month - old Ccp1 - / - Ccp6 - / - mice is significantly decreased ( mean ± SEM ; Mann - Whitney t - test ) .

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Quantification of the thickness of motor and somatosensory cortex of 1- and 5-month-old mice shown in Appendix Fig. S2. The single values shown in Appendix Fig. S2B were averaged, and values from each genotype-specific group were tested against the average values of the remaining three genotypes (Appendix Fig. S2C). Only the cortical thickness of 5-month-old Ccp1-/-Ccp6-/- mice is significantly decreased (mean ±SEM; Mann-Whitney t-test).


Immunohistochemistry of brain sections with anti - MAP2 antibody for visualization of neurons ( layer V ) and apical dendritic ( layer IV ) and with anti - GFAP for reactive astrocytes ( layer V ) . The complete analysis is shown in Fig . EV2A , EV3A . Scale bars : 50 µm . Quantifications of number of MAP2 - positive neurons in layer V , and of number of apical dendrites in layer IV ( Fig . EV2B ) reveal reduced neuron number in layer V , and reduced dendritic density in layer IV of 5 - month - old Ccp1 - / - Ccp6 - / - mice . Quantification of the number of reactive astrocytes in layer V ( Fig . EV3B ) shows a significant increase over wild type in Ccp1 - / - and in Ccp6 - / - mice , however a much stronger increase is seen in Ccp1 - / - Ccp6 - / - mice . Complete analyses are shown in Fig . EV2C , EV3C ( mean ± SEM ; student ' s t - test ) .

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Immunohistochemistry of brain sections with anti-MAP2 antibody for visualization of neurons (layer V) and apical dendritic (layer IV) and with anti-GFAP for reactive astrocytes (layer V). The complete analysis is shown in Fig. EV2A, EV3A. Scale bars: 50 µm. Quantifications of number of MAP2-positive neurons in layer V, and of number of apical dendrites in layer IV (Fig. EV2B) reveal reduced neuron number in layer V, and reduced dendritic density in layer IV of 5-month-old Ccp1-/-Ccp6-/- mice. Quantification of the number of reactive astrocytes in layer V (Fig. EV3B) shows a significant increase over wild type in Ccp1-/- and in Ccp6-/- mice, however a much stronger increase is seen in Ccp1-/-Ccp6-/- mice. Complete analyses are shown in Fig. EV2C, EV3C (mean ±SEM; student's t-test).


Immunohistochemistry of brain sections stained with SMI - 32 antibody ( a representative section of one brain per genotype group is shown , complete analysis in Appendix Fig . S3 ) . Neuronal SMI - 32 labelling indicates increased axonal damage in the cortex of Ccp1 - / - Ccp6 - / - mice at 5 months . Scale bar : 100 µm .

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Immunohistochemistry of brain sections stained with SMI-32 antibody (a representative section of one brain per genotype group is shown, complete analysis in Appendix Fig. S3). Neuronal SMI-32 labelling indicates increased axonal damage in the cortex of Ccp1-/-Ccp6-/- mice at 5 months. Scale bar: 100 µm.


Representative electron microscopy images of sections of wild type and Ccp1 - / - Ccp6 - / - cerebral cortices with myelinated axons . In Ccp1 - / - Ccp6 - / - sections , several myelinated axons show signs of degeneration , such as accumulation of cellular organelles and cytoplasmic densification ( arrows ) , or axonal lysis ( stars ) . Note that only some axons in the observed sections displayed these phenotypes Ccp1 - / - Ccp6 - / - , while such figures were never observed in wild type cortices . Scale bar : 1 µm .

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

Representative electron microscopy images of sections of wild type and Ccp1-/-Ccp6-/- cerebral cortices with myelinated axons. In Ccp1-/-Ccp6-/- sections, several myelinated axons show signs of degeneration, such as accumulation of cellular organelles and cytoplasmic densification (arrows), or axonal lysis (stars). Note that only some axons in the observed sections displayed these phenotypes Ccp1-/-Ccp6-/-, while such figures were never observed in wild type cortices. Scale bar: 1 µm.


Fate of Purkinje cells in 1 - month - old wild type , spastin - / - , Ccp1 - / - and Ccp1 - / - spastin - / - mice visualized with anti - calbindin antibody . Ccp1 - / - and Ccp1 - / - spastin - / - mice show almost complete loss of the Purkinje - cell layer . Scale bar : 500 µm .

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

Fate of Purkinje cells in 1-month-old wild type, spastin-/-, Ccp1-/- and Ccp1-/-spastin-/- mice visualized with anti-calbindin antibody. Ccp1-/- and Ccp1-/-spastin-/- mice show almost complete loss of the Purkinje-cell layer. Scale bar: 500 µm.


Nissl staining shows the complete loss of Purkinje cells in Ccp1 - / - and Ccp1 - / - spastin - / - mice . Scale bar : 20 µm . Purkinje cell layer ( PC ) and granule cell layer ( GC ) are indicated .

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

Nissl staining shows the complete loss of Purkinje cells in Ccp1-/- and Ccp1-/-spastin-/- mice. Scale bar: 20 µm. Purkinje cell layer (PC) and granule cell layer (GC) are indicated.


Calbindin staining of sections showing axons of Purkinje cells as in ( A ) . While wild type and spastin - / - show normal axons , Ccp1 - / - and Ccp1 - / - spastin - / - mice show axonal swellings ( arrow heads ) in the remaining Purkinje cells . Scale bars : 20 µm .

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

Calbindin staining of sections showing axons of Purkinje cells as in (A). While wild type and spastin-/- show normal axons, Ccp1-/- and Ccp1-/-spastin-/- mice show axonal swellings (arrow heads) in the remaining Purkinje cells. Scale bars: 20 µm.


Immunoblot of cell extracts from Ccp1flox / floxCcp6flox / flox hippocampal neurons without , or after transduction with lentivirus expressing either GFP or GFP - 2A - cre after 4 days in culture . Note the specific increase of polyglutamylation ( polyE ) in neurons transduced with GFP - 2A - cre lentivirus .

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

Immunoblot of cell extracts from Ccp1flox/floxCcp6flox/flox hippocampal neurons without, or after transduction with lentivirus expressing either GFP or GFP-2A-cre after 4 days in culture. Note the specific increase of polyglutamylation (polyE) in neurons transduced with GFP-2A-cre lentivirus.


All kymographs of one representative set of experiments . Mitochondria movements ( Movie S1 ) were plotted in kymographs and analyzed as described in ( A ) . Scale bars : 60 sec , 20 µm . Analyses of ( D ) run length and ( E ) speed distribution , represented as scatter dot plots with each point representing a single run extracted from the kymographs shown in ( C ) . Each group represents the pool of measurement of multiple neurons made in the experiment . The black line indicates the median of the distribution with whiskers at interquartile range . Numerical value of the medians is indicated below each plot . ( F ) Overall motility of mitochondria represented as fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s . Values ( below plots ) from one set of experiment ( C ) show a decrease of about 50 % in Ccp1 - / - Ccp6 - / - conditions , while ( G ) mitochondria density is unaltered . ( H - K ) Statistical analyses of mitochondria transport parameters of experiments shown in ( D - G ; Fig . EV4A - D ) . Each bar represents the mean ( ± SEM ) of medians of single experiments for run length ( H ) and speed ( I ) distributions , and average ( ± SEM ) mitochondria motility ( J ) and density ( K ) of all seven experimental sets . Values of fold change between control and Ccp1 - / - Ccp6 - / - conditions are indicated below the graphs . Green bars are for anterograde , and red for retrograde transport events . Significance was tested using unpaired t - test . Average run length ( H ) as well as speed ( I ) showed no significant differences in these two parameters between control and Ccp1 - / - Ccp6 - / - conditions . ( J ) The average of overall motility reveals an about 50 % decrease in overall mitochondria motility , while mitochondria density ( K ) is unaltered between control and Ccp1 - / - Ccp6 - / - conditions .

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

All kymographs of one representative set of experiments. Mitochondria movements (Movie S1) were plotted in kymographs and analyzed as described in (A). Scale bars: 60 sec, 20 µm. Analyses of (D) run length and (E) speed distribution, represented as scatter dot plots with each point representing a single run extracted from the kymographs shown in (C). Each group represents the pool of measurement of multiple neurons made in the experiment. The black line indicates the median of the distribution with whiskers at interquartile range. Numerical value of the medians is indicated below each plot. (F) Overall motility of mitochondria represented as fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s. Values (below plots) from one set of experiment (C) show a decrease of about 50% in Ccp1-/-Ccp6-/- conditions, while (G) mitochondria density is unaltered. (H-K) Statistical analyses of mitochondria transport parameters of experiments shown in (D-G; Fig. EV4A-D). Each bar represents the mean (±SEM) of medians of single experiments for run length (H) and speed (I) distributions, and average (±SEM) mitochondria motility (J) and density (K) of all seven experimental sets. Values of fold change between control and Ccp1-/-Ccp6-/- conditions are indicated below the graphs. Green bars are for anterograde, and red for retrograde transport events. Significance was tested using unpaired t-test. Average run length (H) as well as speed (I) showed no significant differences in these two parameters between control and Ccp1-/-Ccp6-/- conditions. (J) The average of overall motility reveals an about 50% decrease in overall mitochondria motility, while mitochondria density (K) is unaltered between control and Ccp1-/-Ccp6-/- conditions.


( A ) Huh7 . 5 cells were pre - incubated with neutralizing antibody to ACE2 and SARS - CoV - 2 pseudovirus was pre - incubated with patient isolated mAb COVA1 - 18 , COVA1 - 21 and COVA2 - 15 ( 10 µg / mL ) or UF heparin ( 250 IU / mL ) for 30 min at 37 ° C . SARS - CoV - 2 pseudovirus alone or with blocks was added to the cells for 4h at 4 ° C and binding was determined by ELISA . Data information : Data show the mean values and error bars are the SEM . Statistical analysis was performed using ordinary one - way ANOVA with Tukey multiple - comparison test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 ( n = 3 )

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

(A) Huh7.5 cells were pre-incubated with neutralizing antibody to ACE2 and SARS-CoV-2 pseudovirus was pre-incubated with patient isolated mAb COVA1-18, COVA1-21 and COVA2-15 (10 µg/mL) or UF heparin (250 IU/mL) for 30 min at 37°C. SARS-CoV-2 pseudovirus alone or with blocks was added to the cells for 4h at 4°C and binding was determined by ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using ordinary one-way ANOVA with Tukey multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001(n=3)


( D ) Control and EXT1 - / - XG1 cells were exposed to SARS - CoV - 2 pseudovirus or SARS - CoV - 2 pseudovirus pre - treated with 250 IU / mL UF heparin for 30 min at 37 ° C . After incubation for 4h at 4 ° C , cells were lysed and binding was measured by ELISA . Data information : Data show the mean values and error bars are the SEM . Statistical analysis was performed using two - way ANOVA with Dunnett ' s multiple - comparison test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 ( n = 3 ) .

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

(D) Control and EXT1-/- XG1 cells were exposed to SARS-CoV-2 pseudovirus or SARS-CoV-2 pseudovirus pre-treated with 250 IU/mL UF heparin for 30 min at 37°C. After incubation for 4h at 4°C, cells were lysed and binding was measured by ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using two-way ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n=3).


( B ) SARS - CoV - 2 pseudovirus was pre - incubated for 30 min at 37 ° C with UF heparin ( 250 IU / mL ) or LMWHs tinzaparin ( 250 IU / mL ) or dalteparin ( 250 IU / mL ) or LMWH enoxaparin ( 250 IU / mL ) or nadroparin ( 250 IU / mL ) . Huh7 . 5 were exposed to the SARS - CoV - 2 pseudovirus , alone or treated with different LMWHs for 4 hours at 4 ° C , washed , lysed and binding was determined by ELISA . ( C ) SARS - CoV - 2 pseudovirus was pre - incubated for 30 min at 37 ° C with UF heparin ( 250 IU / mL ) or LMWHs tinzaparin ( 250 IU / mL ) or dalteparin ( 250 IU / mL ) or LMWH enoxaparin ( 250 IU / mL ) or nadroparin ( 250 IU / mL ) . Huh7 . 5 cells were infected with SARS - CoV - 2 pseudovirus in presence or absence of different LMWHsSARS - CoV - 2 and infection was determined after 5 days by luciferase reporter activity . Data information : Data show the mean values and error bars are the SEM . Statistical analysis was performed using ( B ) ordinary one - way ANOVA with Tukey ' s multiple - comparison test ( n = 3 donors measured in monoplo ) , ( C ) ordinary one - way ANOVA with Dunnett ' s multiple - comparison test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ( n = 3 donors measured in triplicate )

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SARS-CoV-2 pseudovirus was pre-incubated for 30 min at 37°C with UF heparin (250 IU/mL) or LMWHs tinzaparin (250 IU/mL) or dalteparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) or nadroparin (250 IU/mL). Huh7.5 were exposed to the SARS-CoV-2 pseudovirus, alone or treated with different LMWHs for 4 hours at 4°C, washed, lysed and binding was determined by ELISA. (C) SARS-CoV-2 pseudovirus was pre-incubated for 30 min at 37°C with UF heparin (250 IU/mL) or LMWHs tinzaparin (250 IU/mL) or dalteparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) or nadroparin (250 IU/mL). Huh7.5 cells were infected with SARS-CoV-2 pseudovirus in presence or absence of different LMWHsSARS-CoV-2 and infection was determined after 5 days by luciferase reporter activity. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (B) ordinary one-way ANOVA with Tukey's multiple-comparison test (n=3 donors measured in monoplo), (C) ordinary one-way ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n=3 donors measured in triplicate)


( C , D ) SARS - CoV - 2 binding was measured in polarized Caco - 2 ( C ) and Calu - 3 ( D ) cells in presence or absence of antibodies against ACE2 ( cell incubation ) , and UF heparin ( 250 IU / mL ) or LMWH enoxaparin ( 250 IU / mL ) ( virus incubation ) . Data information : Data show the mean values and error bars are the SEM . Statistical analysis was performed using ordinary one - way ANOVA with Tukey ' s multiple - comparison test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ( C ) ( n = 3 Caco - 2 donors measured in triplicate ) , ( D ) ( n = 3 Calu - 3 donors measured in triplicate )

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

(C, D) SARS-CoV-2 binding was measured in polarized Caco-2 (C) and Calu-3 (D) cells in presence or absence of antibodies against ACE2 (cell incubation), and UF heparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) (virus incubation). Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (C) (n=3 Caco-2 donors measured in triplicate), (D)(n=3 Calu-3 donors measured in triplicate)


( A ) Namalwa cells ectopically expressing either Syndecan 1 or 4 were exposed to either SARS - CoV - 2 pseudovirus alone or SARS - Cov - 2 pseudovirus pre - treated with UF heparin ( 250 IU / mL ) or LMWH enoxaparin ( 250 IU / mL ) for 30 min at 37 ° C . Binding was measured after 4 hours at 4 ° C by ELISA . ( B ) SARS - CoV - 2 isolate ( hCoV - 19 / Italy ) was pre - incubated with LMWH enoxaparin ( 250 IU / mL ) for 30 min at 37 ° C . Namalwa cells expressing Syndecan 1 and 4 were exposed to either SARS - CoV - 2 isolate ( 100 TCID / mL ) or SARS - CoV - 2 isolate ( 100 TCID / mL ) pre - treated with LMWH LMWH enoxaparin ( 250 IU / mL ) for 4 hours at 4 ° C and binding was determined by quantitative real - time PCR . Data show the mean values and error bars are the SEM . Statistical analysis was performed using ( A ) 2way - ANOVA with Dunnett ' s multiple - comparison test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 ( n = 7 ) , ( B ) 2way - ANOVA with Sidak ' s multiple - comparison test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ( n = 3 measured in triplicate ) .

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

(A) Namalwa cells ectopically expressing either Syndecan 1 or 4 were exposed to either SARS-CoV-2 pseudovirus alone or SARS-Cov-2 pseudovirus pre-treated with UF heparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Binding was measured after 4 hours at 4°C by ELISA. (B) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-incubated with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Namalwa cells expressing Syndecan 1 and 4 were exposed to either SARS-CoV-2 isolate (100 TCID/mL) or SARS-CoV-2 isolate (100 TCID/mL) pre-treated with LMWH LMWH enoxaparin (250 IU/mL) for 4 hours at 4°C and binding was determined by quantitative real-time PCR. Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) 2way-ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n = 7), (B) 2way-ANOVA with Sidak's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n = 3 measured in triplicate).


( B ) SARS - CoV - 2 isolate ( hCoV - 19 / Italy ) was pre - treated for 30 min at 37 ° C with either LMWH enoxaparin ( 250 IU / mL ) or one of the following neutralizing antibodies ( COVA1 - 18 , 1 - 21 and 2 - 15 ) , a non - neutralizing antibody ( COVA1 - 27 ) or a human IgG1 isotype control , all at the concentration of 1 pg / mL . SARS - CoV - 2 isolate alone or with blocks was added at a concentration of 100 TICD / mL . Detection of virus binding to Syndecan 1 expressing Namalwa was measured by quantitative real - time PCR . Data information : Data show the mean values and error bars are the SEM . Statistical analysis was performed using ordinary one - way with Tukey ' s multiple - comparison test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 ( n = 2 measured in duplicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-treated for 30 min at 37°C with either LMWH enoxaparin (250 IU/mL) or one of the following neutralizing antibodies (COVA1-18, 1-21 and 2-15), a non-neutralizing antibody (COVA1-27) or a human IgG1 isotype control, all at the concentration of 1 pg/mL. SARS-CoV-2 isolate alone or with blocks was added at a concentration of 100 TICD/mL. Detection of virus binding to Syndecan 1 expressing Namalwa was measured by quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using ordinary one-way with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01 (n=2 measured in duplicates).


( F , G ) DCs ( F ) and LCs ( G ) were pre - incubated with SARS - CoV - 2 pseudovirus for 4 hours at 37 ° C in presence or absence of UF heparin ( 250 IU / mL ) or LMWH enoxaparin ( 250 IU / mL ) , extensively washed and co - cultured with Huh7 . 5 cells . Transmission by DCs or LCs to Huh7 . 5 cells was determined by luciferase reporter activity . Data information : Data show the mean values and error bars are the SEM . ( F ) ordinary one - way ANOVA with Dunnett ' s multiple - comparison test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 ( n = 4 measured in triplicate ) , ( G ) ordinary one - way ANOVA with Tukey ' s multiple - comparison test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ( n = 3 measured in triplicate ) . DCs : Dendritic cells , LCs : Langerhans cells , RLU : relative light

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

(F, G) DCs (F) and LCs (G) were pre-incubated with SARS-CoV-2 pseudovirus for 4 hours at 37°C in presence or absence of UF heparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL), extensively washed and co-cultured with Huh7.5 cells. Transmission by DCs or LCs to Huh7.5 cells was determined by luciferase reporter activity. Data information: Data show the mean values and error bars are the SEM. (F) ordinary one-way ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n=4 measured in triplicate), (G) ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n=3 measured in triplicate). DCs: Dendritic cells, LCs: Langerhans cells, RLU: relative light units


( H , I ) SARS - CoV - 2 isolate ( hCoV - 19 / Italy ) was pre - treated with LMWH enoxaparin ( 250 IU / mL ) for 30 min at 37 ° C . DCs ( H ) and LCs ( I ) were exposed to either the untreated or pre - treated SARS - CoV - 2 isolate ( 100 TCID / mL ) for 24h , washed thoroughly and co - cultured with Huh7 . 5 cells . Quantification of viral RNA was measured by quantitative real - time PCR . Data information : Data show the mean values and error bars are the SEM . ( H , I ) ordinary one - way ANOVA with Tukey ' s multiple - comparison test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ( H ) ( n = 3 in duplicates ) , ( I ) ( n = 4 in duplicates ) . DCs : Dendritic cells , LCs : Langerhans

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

(H, I) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-treated with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. DCs (H) and LCs (I) were exposed to either the untreated or pre-treated SARS-CoV-2 isolate (100 TCID/mL) for 24h, washed thoroughly and co-cultured with Huh7.5 cells. Quantification of viral RNA was measured by quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. (H, I) ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (H) (n=3 in duplicates), (I) (n=4 in duplicates). DCs: Dendritic cells, LCs: Langerhans cells


( B ) ACE2 , Syndecan 1 and Syndecan 4 cell surface expression on nasal epithelial cells , compared to polarized epithelial Calu - 3 was confirmed by quantitative real - time PCR . ( C , D ) Nasal epithelial cells were exposed to SARS - CoV - 2 isolate ( hCoV - 19 / Italy , 100 TCID / mL ) either directly or after pre - treatment with antibodies against ACE2 cell surface receptors ( 1 hour at 37 ° C ) or after pre - treatment with LMWH enoxaparin ( 250 IU / mL ) for 30 min at 37 ° C . Detection of viral binding after 4 hours at 4 ° C ( C ) and persistently - infected cells lysed after 24 hours at 37 ° C ( D ) was determined by quantitative real - time PCR . Data information : Data show the mean values and error bars are the SEM . Statistical analysis was performed using ( C , D ) ordinary on - way ANOVA with Tukey ' s multiple - comparison test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ( C ) ( n = 3 ) , ( D ) ( n = 3 in duplicates ) .

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(B) ACE2, Syndecan 1 and Syndecan 4 cell surface expression on nasal epithelial cells, compared to polarized epithelial Calu-3 was confirmed by quantitative real-time PCR. (C, D) Nasal epithelial cells were exposed to SARS-CoV-2 isolate (hCoV-19/Italy, 100 TCID/mL) either directly or after pre-treatment with antibodies against ACE2 cell surface receptors (1 hour at 37°C )or after pre-treatment with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Detection of viral binding after 4 hours at 4°C (C) and persistently-infected cells lysed after 24 hours at 37°C (D) was determined by quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (C, D) ordinary on-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (C) (n=3), (D) (n=3 in duplicates).


( A ) Chromosome 7 of S . bayanus is incompatible with the S . cerevisiae genome . The chromosome 7 replacement stain ( Sc + Sb - chr7 ) exhibited serious growth defects when grown on the non - fermentable carbon source ( glycerol ) . The respiration defects could be partially rescued when a whole set of S . bayanus chromosomes were provided ( Sc + Sb - chr7 x Sb - ρ⁰ ) , or fully rescued with the addition of S . bayanus mtDNA ( Sc + Sb - chr7 - ρ⁰ x Sb ) . Cell cultures were serially - diluted and spotted onto YPD ( glucose ) or YPG ( glycerol ) plates . The plates were then incubated at 28 ° C until colonies were easily observed . Sc , S . cerevisiae . Sb , S . bayanus . ρ⁰ , mtDNA - less strains .

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(A) Chromosome 7 of S. bayanus is incompatible with the S. cerevisiae genome. The chromosome 7 replacement stain (Sc+Sb-chr7) exhibited serious growth defects when grown on the non-fermentable carbon source (glycerol). The respiration defects could be partially rescued when a whole set of S. bayanus chromosomes were provided (Sc+Sb-chr7 x Sb-ρ⁰), or fully rescued with the addition of S. bayanus mtDNA (Sc+Sb-chr7-ρ⁰ x Sb). Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed. Sc, S. cerevisiae. Sb, S. bayanus. ρ⁰, mtDNA-less strains.


( B ) Ectopically - expressing Sc - CCM1 rescues the respiration defect of Sc + Sb - chr7 . Spot assays for the wild - type S . cerevisiae ( Sc ) and the chromosome 7 replacement stain ( Sc + Sb - chr7 ) carrying an empty plasmid ( + vector ) or the plasmid with the S . cerevisiae CCM1 gene ( + Sc - CCM1 ) .

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(B) Ectopically-expressing Sc-CCM1 rescues the respiration defect of Sc+Sb-chr7. Spot assays for the wild-type S. cerevisiae (Sc) and the chromosome 7 replacement stain (Sc+Sb-chr7) carrying an empty plasmid (+ vector) or the plasmid with the S. cerevisiae CCM1 gene (+ Sc-CCM1).


( C ) Sb - CCM1 incompatibility could only be rescued in the presence of S . bayanus mtDNA . The native CCM1 ORF in S . cerevisiae was replaced by either Sb - CCM1 ( Sc + Sb - CCM1 ) or Sc - CCM1 ( Sc + Sc - CCM1 , as a control ) coding regions . Crossing the Sc + Sb - CCM1 strain with the mtDNA - less Sb - ρ⁰ strain ( Sc + Sb - CCM1 x Sb - ρ⁰ ) could not rescue the growth defect .

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(C) Sb-CCM1 incompatibility could only be rescued in the presence of S. bayanus mtDNA. The native CCM1 ORF in S. cerevisiae was replaced by either Sb-CCM1 (Sc+Sb-CCM1) or Sc-CCM1 (Sc+Sc-CCM1, as a control) coding regions. Crossing the Sc+Sb-CCM1 strain with the mtDNA-less Sb-ρ⁰ strain (Sc+Sb-CCM1 x Sb-ρ⁰) could not rescue the growth defect.


( D ) CCM1 incompatibility is symmetric between S . cerevisiae and S . bayanus . The CCM1 ORF in S . bayanus was replaced by either Sc - CCM1 ( Sb + Sc - CCM1 ) or Sb - CCM1 ( Sb + Sb - CCM1 , as a control ) coding regions . Only the Sb + Sc - CCM1 strain exhibited respiration defects and the defect could not be rescued even when a whole set of S . cerevisiae chromosomes were provided ( Sb + Sc - CCM1 x Sc - ρ⁰ ) .

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(D) CCM1 incompatibility is symmetric between S. cerevisiae and S. bayanus. The CCM1 ORF in S. bayanus was replaced by either Sc-CCM1 (Sb+Sc-CCM1) or Sb-CCM1 (Sb+Sb-CCM1, as a control) coding regions. Only the Sb+Sc-CCM1 strain exhibited respiration defects and the defect could not be rescued even when a whole set of S. cerevisiae chromosomes were provided (Sb+Sc-CCM1 x Sc-ρ⁰).


( E ) CCM1 incompatibility probably occurred during the divergence between S . bayanus and the common ancestor of S . cerevisiae , S . paradoxus , S . mikatae and S . kudriavzevii . The endogenous CCM1 in S . cerevisiae was replaced with its orthologous alleles from other Saccharomyces sensu stricto yeasts . Only the strain carrying S . bayanus CCM1 displayed growth defects on glycerol - containing plates .

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(E) CCM1 incompatibility probably occurred during the divergence between S. bayanus and the common ancestor of S. cerevisiae, S. paradoxus, S. mikatae and S. kudriavzevii. The endogenous CCM1 in S. cerevisiae was replaced with its orthologous alleles from other Saccharomyces sensu stricto yeasts. Only the strain carrying S. bayanus CCM1 displayed growth defects on glycerol-containing plates.


( A ) Sb - Ccm1 protein is transported efficiently to S . cerevisiae mitochondria . Total cell extracts ( total ) , cytosolic fractions ( cyto ) or mitochondrial fractions ( mito ) were hybridized with different antibodies to detect the proteins . G6PDH ( glucose - 6 - phosphate dehydrogenase ) and Cox2 served as cytosolic and mitochondrial markers , respectively . Both Sc - Ccm1 and Sb - Ccm1 were fused with c - Myc at the C - terminus and expressed from a single - copy plasmid . The Myc - tagged Ccm1 proteins consistently appeared as double bands , which were probably due to some unknown protein modifications .

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(A) Sb-Ccm1 protein is transported efficiently to S. cerevisiae mitochondria. Total cell extracts (total), cytosolic fractions (cyto) or mitochondrial fractions (mito) were hybridized with different antibodies to detect the proteins. G6PDH (glucose-6-phosphate dehydrogenase) and Cox2 served as cytosolic and mitochondrial markers, respectively. Both Sc-Ccm1 and Sb-Ccm1 were fused with c-Myc at the C-terminus and expressed from a single-copy plasmid. The Myc-tagged Ccm1 proteins consistently appeared as double bands, which were probably due to some unknown protein modifications.


( B ) The level of 15S rRNA is reduced in Sc + Sb - CCM1 cells . RNA isolated from Sc + Sb - CCM1 ( labeled as Sb ) or Sc + Sc - CCM1 ( labeled as Sc ) cells was hybridized with gene - specific probes using Northern blots .

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(B) The level of 15S rRNA is reduced in Sc+Sb-CCM1 cells. RNA isolated from Sc+Sb-CCM1 (labeled as Sb) or Sc+Sc-CCM1 (labeled as Sc) cells was hybridized with gene-specific probes using Northern blots.


( C ) Sb - Ccm1 interacts weakly with S . cerevisiae 15S rRNA . The level of Ccm1 - bound 15S rRNA was measured using quantitative PCR analysis following mitochondrial RNA immunoprecipitation of S . cerevisiae cells expressing Sc - CCM1 - 13Myc or Sb - CCM1 - 13Myc . Data was normalized to the control group without the Myc tag to obtain the relative fold enrichment . Graph was plotted using data from three independent repeats for each strain and p value was calculated by unpaired , two - sided student t - test . Error bars indicate S . D . * * * , p value < 0 . 001 .

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(C) Sb-Ccm1 interacts weakly with S. cerevisiae 15S rRNA. The level of Ccm1-bound 15S rRNA was measured using quantitative PCR analysis following mitochondrial RNA immunoprecipitation of S. cerevisiae cells expressing Sc-CCM1-13Myc or Sb-CCM1-13Myc. Data was normalized to the control group without the Myc tag to obtain the relative fold enrichment. Graph was plotted using data from three independent repeats for each strain and p value was calculated by unpaired, two-sided student t-test. Error bars indicate S.D. ***, p value < 0.001.


( D ) Overexpressing Sb - CCM1 rescues the respiration defect of Sc + Sb - CCM1 cells . Cell cultures were serially - diluted and spotted onto YPD ( glucose ) or YPG ( glycerol ) plates . The plates were then incubated at 28 ° C until colonies were easily observed .

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(D) Overexpressing Sb-CCM1 rescues the respiration defect of Sc+Sb-CCM1 cells. Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed.


( E ) The steady - state level of mtDNA - encoded proteins is reduced in Sc + Sb - CCM1 cells . Immunoblotting for the mitochondrial proteins in Sc + Sb - CCM1 ( labeled as Sb ) or Sc + Sc - CCM1 ( labeled as Sc ) cells . Cox1 , Cox2 and Cox3 are mtDNA - encoded complex IV subunits . Tom20 is a nucleus - encoded mitochondrial protein .

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(E) The steady-state level of mtDNA-encoded proteins is reduced in Sc+Sb-CCM1 cells. Immunoblotting for the mitochondrial proteins in Sc+Sb-CCM1 (labeled as Sb) or Sc+Sc-CCM1 (labeled as Sc) cells. Cox1, Cox2 and Cox3 are mtDNA-encoded complex IV subunits. Tom20 is a nucleus-encoded mitochondrial protein.


( A ) Tetrad analysis reveals that the respiration defect of Sc + Sb - CCM1 cells can be rescued by mutations in mitochondrial or nuclear genomes . The suppressor clones were crossed with the Sc + Sb - CCM1 - ρ⁰ strain and the tetrads from the cross were analyzed . A non - Mendelian 4 : 0 segregation pattern for improved respiration is expected if the suppressor mutation is in mtDNA ( left panel ) . Otherwise , a 2 : 2 segregation pattern would be observed if the rescue effect involved a single nuclear mutation ( right panel ) .

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(A) Tetrad analysis reveals that the respiration defect of Sc+Sb-CCM1 cells can be rescued by mutations in mitochondrial or nuclear genomes. The suppressor clones were crossed with the Sc+Sb-CCM1-ρ⁰ strain and the tetrads from the cross were analyzed. A non-Mendelian 4:0 segregation pattern for improved respiration is expected if the suppressor mutation is in mtDNA (left panel). Otherwise, a 2:2 segregation pattern would be observed if the rescue effect involved a single nuclear mutation (right panel).


( A ) Reconstituted Sb - CCM1 mutants can rescue the incompatibility of Sb - CCM1 . Among 94 suppressor clones , 20 of them carry mutations in the Sb - CCM1 gene . These mutants were reconstructed in Sc + Sb - CCM1 cells to test their function . Superscripts next to Sb - CCM1 indicate the respective amino acid substitutions and numbers in parentheses indicate the number of independent clones in our suppressor screen . Sc , wild - type S . cerevisiae .

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(A) Reconstituted Sb-CCM1 mutants can rescue the incompatibility of Sb-CCM1. Among 94 suppressor clones, 20 of them carry mutations in the Sb-CCM1 gene. These mutants were reconstructed in Sc+Sb-CCM1 cells to test their function. Superscripts next to Sb-CCM1 indicate the respective amino acid substitutions and numbers in parentheses indicate the number of independent clones in our suppressor screen. Sc, wild-type S. cerevisiae.


( C ) The 400th residue plays a determinant role in the S . cerevisiae - S . bayanus incompatibility . Both Sb - CCM1E375Q and Sb - CCM1D400N mutants relieved the incompatibility in S . cerevisiae , but only Sb - CCM1D400N caused respiration defects in S . bayanus . The Sb - CCM1E375Q mutant was selected as a control in this experiment since it was a charged - to - polar substitution similar to Sb - CCM1D400N and also appeared three times in our screen . Sb , wild - type S . bayanus .

(C) The 400th residue plays a determinant role in the S. cerevisiae-S. bayanus incompatibility. Both Sb-CCM1E375Q and Sb-CCM1D400N mutants relieved the incompatibility in S. cerevisiae, but only Sb-CCM1D400N caused respiration defects in S. bayanus. The Sb-CCM1E375Q mutant was selected as a control in this experiment since it was a charged-to-polar substitution similar to Sb-CCM1D400N and also appeared three times in our screen. Sb, wild-type S. bayanus.


( D ) Protein structural analysis shows that all intramolecular suppressor mutations were located on the predicted PPR motifs . The predicted protein structure of Ccm1 is shown at left . Blue and green colors indicate the two α - helices in each predicted PPR motif and white color indicates additional α - helices in the N - terminus and the middle region . Red color indicates the positions of the suppressor mutations . Predicted PPR motifs occupy around 52 % of the whole protein ( 453 of 871 residues ) . The box at right displays a magnified version of some PPR motifs where the substitutions are located .

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(D) Protein structural analysis shows that all intramolecular suppressor mutations were located on the predicted PPR motifs. The predicted protein structure of Ccm1 is shown at left. Blue and green colors indicate the two α-helices in each predicted PPR motif and white color indicates additional α-helices in the N-terminus and the middle region. Red color indicates the positions of the suppressor mutations. Predicted PPR motifs occupy around 52% of the whole protein (453 of 871 residues). The box at right displays a magnified version of some PPR motifs where the substitutions are located.


( A ) 15S rRNA levels are increased in the suppressor clones . Total RNA was isolated from the replacement strains ( Sc + Sc - CCM1 and Sc + Sb - CCM1 ) , two nuclear suppressor strains ( Sc + Sb - CCM1D400N and Sc + Sb - CCM1E375Q ) and one mitochondrial suppressor strain and then subjected to quantitative PCR analyses . Relative mRNA abundance was calculated by normalizing the 15S rRNA level to the ATP6 mRNA level . Graph was plotted using data from five independent repeats for each strain and p values were calculated by unpaired , two - sided student t - test . Error bars indicate S . D . * * * , p value < 0 . 001 . * * , p value < 0 . 01 . Asterisks in black , comparing Sc + Sc - CCM1 with others . Asterisks in gray , comparing Sc + Sb - CCM1 with others .

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(A) 15S rRNA levels are increased in the suppressor clones. Total RNA was isolated from the replacement strains (Sc+Sc-CCM1 and Sc+Sb-CCM1), two nuclear suppressor strains (Sc+Sb-CCM1D400N and Sc+Sb-CCM1E375Q) and one mitochondrial suppressor strain and then subjected to quantitative PCR analyses. Relative mRNA abundance was calculated by normalizing the 15S rRNA level to the ATP6 mRNA level. Graph was plotted using data from five independent repeats for each strain and p values were calculated by unpaired, two-sided student t-test. Error bars indicate S.D. ***, p value < 0.001. **, p value < 0.01. Asterisks in black, comparing Sc+Sc-CCM1 with others. Asterisks in gray, comparing Sc+Sb-CCM1 with others.


( B ) The steady - state level of mtDNA - encoded proteins is increased in the suppressor clones . Protein extracts from the replacement and suppressor strains were hybridized with antibodies against G6PDH , Cox1 , Cox2 or Cox3 .

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(B) The steady-state level of mtDNA-encoded proteins is increased in the suppressor clones. Protein extracts from the replacement and suppressor strains were hybridized with antibodies against G6PDH, Cox1, Cox2 or Cox3.


( B ) Seven of 12 tested PPR genes exhibit incompatibility between S . cerevisiae and at least one other Saccharomyces sensu stricto species . The doubling time of S . cerevisiae cells carrying the endogenous PPR genes ( Sc ) or orthologs of other yeast species ( Sp , Sm , Sk and Sb ) were measured in YPD ( glucose ) or YPG ( glycerol ) media . All the measurements were then normalized to the doubling time of Sc to obtain the relative doubling time . Graphs were plotted using data from at least three independent repeats for each strain . Sc , S . cerevisiae . Sp , S . paradoxus . Sm , S . mikatae . Sk , S . kudriavzevii . Sb , S . bayanus . n . d . , growth not detected . p values were calculated by unpaired , two - sided student t - test . Error bars indicate S . D . * , p value < 0 . 05 . * * , p value < 0 . 01 . * * * , p value < 0 . 001 .

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

(B) Seven of 12 tested PPR genes exhibit incompatibility between S. cerevisiae and at least one other Saccharomyces sensu stricto species. The doubling time of S. cerevisiae cells carrying the endogenous PPR genes (Sc) or orthologs of other yeast species (Sp, Sm, Sk and Sb) were measured in YPD (glucose) or YPG (glycerol) media. All the measurements were then normalized to the doubling time of Sc to obtain the relative doubling time. Graphs were plotted using data from at least three independent repeats for each strain. Sc, S. cerevisiae. Sp, S. paradoxus. Sm, S. mikatae. Sk, S. kudriavzevii. Sb, S. bayanus. n.d., growth not detected. p values were calculated by unpaired, two-sided student t-test. Error bars indicate S.D. *, p value < 0.05. **, p value < 0.01. ***, p value < 0.001.


Pregnant dams were injected with 4 - OHT at days E10 . 5 and E11 . 5 for analysis at day E12 . 5 or E10 . 5 , E11 . 5 , and E12 . 5 of development for later times of analysis Developmental loss of VE - cadherin leads to severe edema . Fetuses of the genotypes listed in the top row were analyzed at the developmental stages in the first column . Numbers in brackets denote embryos with signs of edema . Last column lists the percentage of viable fetuses in the VE - cadherin deleted cohort Fetuses were explanted at day E14 . 5 . Arrowheads indicate prominent edema along the back . The images are representative for 42 ( Cdh5lox / lox ) and 25 ( Cdh5lox / lox ; Prox1 - CreERT2 ) analyzed

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Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis Developmental loss of VE-cadherin leads to severe edema. Fetuses of the genotypes listed in the top row were analyzed at the developmental stages in the first column. Numbers in brackets denote embryos with signs of edema. Last column lists the percentage of viable fetuses in the VE-cadherin deleted cohort Fetuses were explanted at day E14.5. Arrowheads indicate prominent edema along the back. The images are representative for 42 (Cdh5lox/lox) and 25 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals


Pregnant dams were injected with 4 - OHT at days E10 . 5 and E11 . 5 for analysis at day E12 . 5 or E10 . 5 , E11 . 5 , and E12 . 5 of development for later times of analysis Skin wholemount preparations stained for PROX1 , PECAM1 and VEGFR - 3 . Maximum intensity projections ( MIPs ) of tiled confocal stacks ( Cdh5lox / lox 2075µm x 2075µm , z = 50µm ; Cdh5lox / lox ; Prox1 - CreERT2 3765µm x 3765 µm , z = 61µm ) . Scale bars = 100 μm . The data are representative for 6 ( Cdh5lox / lox ) and 6 ( Cdh5lox / lox ; Prox1 - CreERT2 ) analyzed animals from 3

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Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis Skin wholemount preparations stained for PROX1, PECAM1 and VEGFR-3. Maximum intensity projections (MIPs) of tiled confocal stacks (Cdh5lox/lox 2075µm x 2075µm, z=50µm; Cdh5lox/lox; Prox1-CreERT2 3765µm x 3765 µm, z=61µm). Scale bars = 100 μm. The data are representative for 6 (Cdh5lox/lox) and 6 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 3 litters


Pregnant dams were injected with 4 - OHT at days E10 . 5 and E11 . 5 for analysis at day E12 . 5 or E10 . 5 , E11 . 5 , and E12 . 5 of development for later times of analysis Enumeration of the PROX1 - expressing ( PROX1 + ) nuclei in three different VE - cadherin deleted wholemount preparations analogous to Fig . 1B . Nuclei were counted in a MIP corresponding to an area of 1 . 5 mm2 and normalized to the number of nuclei in Cdh5lox / lox control preparations The area covered by lymphatic vessels was determined in the samples evaluated in ( D ) and is depicted as relative area compared to Cdh5lox / lox controls . The calculations and measurements were obtained from three animals and three confocal stacks per biological group . PROX1 - positive nuclei were counted using the particle analysis tool of

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Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis Enumeration of the PROX1-expressing (PROX1+) nuclei in three different VE-cadherin deleted wholemount preparations analogous to Fig. 1B. Nuclei were counted in a MIP corresponding to an area of 1.5 mm2 and normalized to the number of nuclei in Cdh5lox/lox control preparations The area covered by lymphatic vessels was determined in the samples evaluated in (D) and is depicted as relative area compared to Cdh5lox/lox controls. The calculations and measurements were obtained from three animals and three confocal stacks per biological group. PROX1-positive nuclei were counted using the particle analysis tool of Fiji


Deletion of VE - cadherin in lymphatic endothelial cells of newborn pups was induced by two optimal doses of 4 - OHT at days P2 and P4 . For analysis 6 weeks later ear punches were subjected to wholemount immunostaining of the dermal vasculature . A - H ( A , B , E , F ) Overview MIPs of ear skin wholemount preparations immunostained for the indicated antigens . VEC , VE - cadherin ; PRX , PROX1 . ( C , D , G , H ) Magnified views of the areas in the red boxes in ( A , E ) . Areas delimited by the stippled yellow boxes in ( C ) and ( G ) are enlarged in the insets ( yellow solid boxes ) . Yellow arrows indicate widened lumen , yellow arrowheads denote oak leaf - shaped LECs with discontinuous junctions . Scale bars in ( A and E ) correspond to 100 μm and in ( C and G ) to 50 µm . The data are representative for 6 ( Cdh5lox / lox ) and 5 ( Cdh5lox / lox ; Prox1 - CreERT2 ) analyzed animals from 2 litters

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

Deletion of VE-cadherin in lymphatic endothelial cells of newborn pups was induced by two optimal doses of 4-OHT at days P2 and P4. For analysis 6 weeks later ear punches were subjected to wholemount immunostaining of the dermal vasculature. A-H (A, B, E, F) Overview MIPs of ear skin wholemount preparations immunostained for the indicated antigens. VEC, VE-cadherin; PRX, PROX1. (C, D, G, H) Magnified views of the areas in the red boxes in (A, E). Areas delimited by the stippled yellow boxes in (C) and (G) are enlarged in the insets (yellow solid boxes). Yellow arrows indicate widened lumen, yellow arrowheads denote oak leaf-shaped LECs with discontinuous junctions. Scale bars in (A and E) correspond to 100 μm and in (C and G) to 50 µm. The data are representative for 6 (Cdh5lox/lox) and 5 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 2 litters


A - L VE - cadherin deletion was initiated either in newborn pups ( A , B , E , F ) by two applications of 4 - OHT at days P2 and P4 or in mice at 11 weeks ( C , G ) and 42 weeks ( D , H ) of age ( three applications of Tamoxifen at 2 day intervals via oral gavage ) . Ear skins were prepared 6 weeks ( A , B , E , F ) or 5 weeks ( C , D , G , H ) after induction and immunostained for the indicated proteins . Shown are MIPs of confocal tile scans ( approx . 750 µm x 750 µm ) covering 40 µm in depth . White arrows in ( F ) highlight distorted and partially fragmented lymphatic vessels and white arrowheads denote aberrantly pointed vessels ( F ) . ( I - L ) Lymphatic valves in the dermis of the ear were maintained for 5 weeks despite deletion of VE - cadherin at 11 weeks of age ( yellow arrows in K , L ) . PEC1 , PECAM1 ; PRX1 , PROX1 ; VR3 , VEGFR - 3 . Scale bars correspond to 100 μm . The data represent wholemount stainings from 6 ( A , E ) , 5 ( B , F ) , 6 ( C , G , K , L ) , 3 ( D , H ) and 6 ( I , J ) analyzed animals

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L VE-cadherin deletion was initiated either in newborn pups (A, B, E, F) by two applications of 4-OHT at days P2 and P4 or in mice at 11 weeks (C, G) and 42 weeks (D, H) of age (three applications of Tamoxifen at 2 day intervals via oral gavage). Ear skins were prepared 6 weeks (A, B, E, F) or 5 weeks (C, D, G, H) after induction and immunostained for the indicated proteins. Shown are MIPs of confocal tile scans (approx. 750 µm x 750 µm) covering 40 µm in depth. White arrows in (F) highlight distorted and partially fragmented lymphatic vessels and white arrowheads denote aberrantly pointed vessels (F). (I-L) Lymphatic valves in the dermis of the ear were maintained for 5 weeks despite deletion of VE-cadherin at 11 weeks of age (yellow arrows in K, L). PEC1, PECAM1; PRX1, PROX1;VR3, VEGFR-3. Scale bars correspond to 100 μm. The data represent wholemount stainings from 6 (A, E), 5 (B, F), 6 (C, G, K, L), 3 (D, H) and 6 (I, J) analyzed animals


Fig . 4 : VE - cadherin is indispensable for the maintenance of mesenteric lymphatic vessels at all ages . A - F Deletion of VE - cadherin was induced in mice at various age ( 4 weeks ( A ) , neonate ( B ) , 6 month ( C , D , E ) or 1 year ( F ) ) by either two applications of 4 - OHT at days P2 and P4 ( neonates ) or three applications of Tamoxifen at 2 day intervals via oral gavage ( all other ages ) . The mesentery was prepared as indicated 4 , 6 , 8 or 11 weeks later and the wholemount preparations were immunostained for the proteins specified in color ( left ) . ( B ) Inset in top panel shows a magnification of the area outlined by the white dashed line ( for further magnification see expanded view Fig . 1A and B ) . Arrows indicate the previous position of lymphatic valves ; white arrowheads indicate PECAM1 + capillaries supplying the intestinal fat tissue lining the vessels ( magnified in expanded view Fig . 1C ) ; yellow arrowheads denote island of LECs that display intracellular VEGFR - 3 staining . ( C ) Arrows denote LECs that have converted from growth as tubes to sheets ; white arrowheads , thinning of lymphatic vessels ; yellow arrowheads , aberrant sprouting of LECs . The area outlined by a white box is depicted magnified in ( D ) and provides details on LECs growing as a sheet of cells . ( E ) Formation of ectopic sprouts from the valve area of degenerating lymphatic vessels . ( F ) Fully deteriorated lymphatic vessel with emanating sprouts that initiate sheet - like growth . PEC1 , PECAM1 ; PRX1 , PROX1 ; VR3 , VEGFR - 3 . Scale bars = 100 μm . The data represent n = 5 ( A ) , n = 5 ( B ) , n = 3 ( C - E ) and n = 6 ( F ) animals .

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

Fig. 4: VE-cadherin is indispensable for the maintenance of mesenteric lymphatic vessels at all ages. A-F Deletion of VE-cadherin was induced in mice at various age (4 weeks (A), neonate (B), 6 month (C, D, E) or 1 year (F)) by either two applications of 4-OHT at days P2 and P4 (neonates) or three applications of Tamoxifen at 2 day intervals via oral gavage (all other ages). The mesentery was prepared as indicated 4, 6, 8 or 11 weeks later and the wholemount preparations were immunostained for the proteins specified in color (left). (B) Inset in top panel shows a magnification of the area outlined by the white dashed line (for further magnification see expanded view Fig. 1A and B). Arrows indicate the previous position of lymphatic valves; white arrowheads indicate PECAM1+ capillaries supplying the intestinal fat tissue lining the vessels (magnified in expanded view Fig. 1C); yellow arrowheads denote island of LECs that display intracellular VEGFR-3 staining. (C) Arrows denote LECs that have converted from growth as tubes to sheets; white arrowheads, thinning of lymphatic vessels; yellow arrowheads, aberrant sprouting of LECs. The area outlined by a white box is depicted magnified in (D) and provides details on LECs growing as a sheet of cells. (E) Formation of ectopic sprouts from the valve area of degenerating lymphatic vessels. (F) Fully deteriorated lymphatic vessel with emanating sprouts that initiate sheet-like growth. PEC1, PECAM1; PRX1, PROX1; VR3, VEGFR-3. Scale bars = 100 μm. The data represent n=5 (A), n=5 (B), n=3 (C-E) and n=6 (F) animals.


Fig . 5 : Deletion of VE - cadherin results in fragmentation and distension of the intestinal lacteals . A - E Deletion of VE - cadherin was induced in mice at various ages ( neonatal ( C ) , 16 weeks ( D ) or 1 year ( E ) ) by either two applications of 4 - OHT at days P2 and P4 ( neonates ) or three applications of Tamoxifen at 2 day intervals via oral gavage ( all other ages ) . Control mice of the same age were subjected to an identical regimen ( A , B ) . Intestinal wholemount preparations of the jejunum and ileum were generated 5 - 8 weeks after Tamoxifen induction and immunostained for the indicated proteins , scale bars = 100 μm . The data represent wholemount stainings from 3 ( D ) , 5 ( B , E ) and 6 ( A , C ) animals analyzed for each genotype .

Fig. 5: Deletion of VE-cadherin results in fragmentation and distension of the intestinal lacteals. A-E Deletion of VE-cadherin was induced in mice at various ages (neonatal (C), 16 weeks (D) or 1 year (E)) by either two applications of 4-OHT at days P2 and P4 (neonates) or three applications of Tamoxifen at 2 day intervals via oral gavage (all other ages).Control mice of the same age were subjected to an identical regimen (A, B). Intestinal wholemount preparations of the jejunum and ileum were generated 5 - 8 weeks after Tamoxifen induction and immunostained for the indicated proteins, scale bars = 100 μm. The data represent wholemount stainings from 3 (D), 5 (B,E) and 6 (A, C) animals analyzed for each genotype.


Deletion of VE - cadherin was induced in adult mice ( 13 weeks ) and EdU incorporation into PROX1 - positive cell nuclei was determined 6 weeks after Tamoxifen administration . Shown are mesenteric wholemount stainings prepared from control ( A , B ; n = 3 ) or VE - cadherin - deleted mice ( C , D ; n = 3 ) . ( B ) and ( D ) show a magnification of the area outlined by the white dashed line in ( A ) and ( C ) . Antigens shown in addition to the EdU incorporation are depicted in colour on the left side of the first panel . PEC , PECAM1 ; PRX1 , PROX1 . Scale bars = 100 µm Enumeration of EdU - positive LECs . The measurements were obtained from three animals and nine confocal stacks per biological group . PROX1 - positive nuclei were counted using the particle analysis tool of Fiji . Co - localization of PROX1 and EdU was determined manually in each confocal plane , using the cell counting tool of Fiji . The data represent mean ± SD . * * * * p ≤ 0 . 0001 . Two - tailed unpaired Student ' s t

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

Deletion of VE-cadherin was induced in adult mice (13 weeks) and EdU incorporation into PROX1-positive cell nuclei was determined 6 weeks after Tamoxifen administration. Shown are mesenteric wholemount stainings prepared from control (A, B; n=3) or VE-cadherin-deleted mice (C, D; n=3). (B) and (D) show a magnification of the area outlined by the white dashed line in (A) and (C). Antigens shown in addition to the EdU incorporation are depicted in colour on the left side of the first panel. PEC, PECAM1; PRX1, PROX1. Scale bars = 100 µm Enumeration of EdU-positive LECs. The measurements were obtained from three animals and nine confocal stacks per biological group. PROX1-positive nuclei were counted using the particle analysis tool of Fiji. Co-localization of PROX1 and EdU was determined manually in each confocal plane, using the cell counting tool of Fiji. The data represent mean ± SD. **** p ≤ 0.0001. Two-tailed unpaired Student's t test


Aberrantly enhanced VEGFR - 3 phosphorylation in VE - cadherin - deficient mesenteric LECs of six weeks old mice that had received 4 - OHT injections at P2 and P4 . Shown are wholemount immunostainings for the antigens shown on the left in color . pVR3 , pVEGFR - 3 , PRX1 , PROX1 ; PEC1 , PECAM1 . ( G ) and ( I ) show a magnification of the area outlined by the white dashed box in ( F ) and ( H ) . Scale bars = 50 μm . The data are representative for 6 ( Cdh5lox / lox ) and 5 ( Cdh5lox / lox ; Prox1 - CreERT2 ) analyzed animals from 2

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

Aberrantly enhanced VEGFR-3 phosphorylation in VE-cadherin-deficient mesenteric LECs of six weeks old mice that had received 4-OHT injections at P2 and P4. Shown are wholemount immunostainings for the antigens shown on the left in color. pVR3, pVEGFR-3, PRX1, PROX1; PEC1, PECAM1. (G) and (I) show a magnification of the area outlined by the white dashed box in (F) and (H). Scale bars = 50 μm. The data are representative for 6 (Cdh5lox/lox) and 5 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 2 litters


Dermal , mesenteric and intestinal tissue pieces of 15 week old control ( Cdh5lox / lox ) mice were dissociated and total RNA was extracted immediately . For reverse transcription 2 . 0 µg of total RNA was used for each tissue type . Expression of lymphangiogenic factors VEGF - C and VEGF - D was determined by quantitative real - time ( qRT ) - PCR using specific TaqMan probes . Samples were measured in triplicate and diagrams are representative for 3 independent experiments . Relative expression ( ΔΔCt ) of target genes was normalized to the control transcript

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

Dermal, mesenteric and intestinal tissue pieces of 15 week old control (Cdh5lox/lox) mice were dissociated and total RNA was extracted immediately. For reverse transcription 2.0 µg of total RNA was used for each tissue type. Expression of lymphangiogenic factors VEGF-C and VEGF-D was determined by quantitative real-time (qRT)-PCR using specific TaqMan probes. Samples were measured in triplicate and diagrams are representative for 3 independent experiments. Relative expression (ΔΔCt) of target genes was normalized to the control transcript UBC


Loss of VEGFR - 3 surface expression on mesenteric LECs growing in cell sheets was associated with concomitant upregulation of VEGFR - 2 , scale bars = 50 μm . The data are representative for 4 analyzed animals from 2 litters . VR2 , VEGFR - 2 ; VR3 , VEGFR - 3 , PRX1 ,

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

Loss of VEGFR-3 surface expression on mesenteric LECs growing in cell sheets was associated with concomitant upregulation of VEGFR-2, scale bars = 50 μm. The data are representative for 4 analyzed animals from 2 litters.VR2, VEGFR-2; VR3, VEGFR-3, PRX1, PROX1


Expression of the transcriptional co - activators YAP and TAZ was analyzed in mesenteric wholemount preparations 8 weeks after induction of VE - Cadherin deletion at 1 year of age by three doses of Tamoxifen . Respective antigens depicted are indicated on the top of the panels in colour . The highly related molecules YAP and TAZ were both recognized by the antibody used and are therefore collectively abbreviated Y / T . Scale bars = 100

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

Expression of the transcriptional co-activators YAP and TAZ was analyzed in mesenteric wholemount preparations 8 weeks after induction of VE-Cadherin deletion at 1 year of age by three doses of Tamoxifen. Respective antigens depicted are indicated on the top of the panels in colour. The highly related molecules YAP and TAZ were both recognized by the antibody used and are therefore collectively abbreviated Y/T. Scale bars = 100 μm


Fig . 7 : Loss of VE - cadherin from lymphatic endothelial cells results in increased circumferential actin and a reduced number of focal adhesions . A , B Primary dermal LECs were isolated as described in appendix Fig . S8 A . PdLECs were used for up to four passages and control and VE - cadherin - deleted pdLECs had been cultured for the same number of passages . Cells were seeded at a density of 2 . 5 x 104 cells on gelatine - coated 15mm glass coverslips and co - stained after 3 days of culture for the actin cytoskeleton ( Phalloidin ) and either Vinculin ( A ) or FAK ( B ) for the visualization of focal adhesions . PROX1 staining in ( A ) identifies LECs , nuclei in ( B ) were counterstained with Hoechst . Scale bars correspond to 50 µm . ( C , D ) Bar diagrams show the number of focal adhesions / cell , which were enumerated using Image J from the Vinculin and FAK stainings shown in ( A and B ) . Data are mean values + S . E . M . derived from three cell lines and 9 viewfields of 256 µm x 256 µm per biological group . * * * p ≤ 0 . 001 . Two - tailed unpaired Student ' s t test .

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

Fig. 7: Loss of VE-cadherin from lymphatic endothelial cells results in increased circumferential actin and a reduced number of focal adhesions. A, B Primary dermal LECs were isolated as described in appendix Fig. S8 A. PdLECs were used for up to four passages and control and VE-cadherin-deleted pdLECs had been cultured for the same number of passages. Cells were seeded at a density of 2.5 x 104 cells on gelatine-coated 15mm glass coverslips and co-stained after 3 days of culture for the actin cytoskeleton (Phalloidin) and either Vinculin (A) or FAK (B) for the visualization of focal adhesions. PROX1 staining in (A) identifies LECs, nuclei in (B) were counterstained with Hoechst. Scale bars correspond to 50 µm. (C, D) Bar diagrams show the number of focal adhesions/cell, which were enumerated using Image J from the Vinculin and FAK stainings shown in (A and B). Data are mean values + S.E.M. derived from three cell lines and 9 viewfields of 256 µm x 256 µm per biological group. *** p ≤ 0.001. Two-tailed unpaired Student's t test.


Fig . 8 : Enhanced collective cell migration in VE - cadherin - deficient lymphatic endothelial cells . ( A ) Collective cell migration of VE - cadherin - deficient and control LECs was assessed in wound scratch assays . LECs were grown up to four passages and seeded at a density of 5x104 cells on fibronectin - coated 8 well µ - chamber slides ( growth area 0 . 22 cm2 ) ; scale bar 500 µm ( B ) The area of the zone denuded from cells is depicted in the curve diagram showing mean values ± SD of three individual experiments conducted in quadruplets . * p ≤ 0 . 01 . Two - tailed unpaired Student ' s t test . Expanded View Figures - Legends

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

Fig. 8: Enhanced collective cell migration in VE-cadherin-deficient lymphatic endothelial cells. (A) Collective cell migration of VE-cadherin-deficient and control LECs was assessed in wound scratch assays. LECs were grown up to four passages and seeded at a density of 5x104 cells on fibronectin-coated 8 well µ-chamber slides (growth area 0.22 cm2); scale bar 500 µm (B) The area of the zone denuded from cells is depicted in the curve diagram showing mean values ± SD of three individual experiments conducted in quadruplets. * p ≤ 0.01. Two-tailed unpaired Student's t test. Expanded View Figures - Legends