A Neurosphere assays were performed using mouse E14 . 5 primary neural progenitors treated with CRH with or without antalarmin ( Ant ) ( n = 3 biological replicates ) .

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A Neurosphere assays were performed using mouse E14.5 primary neural progenitors treated with CRH with or without antalarmin (Ant) (n = 3 biological replicates).


B Effects of CRH treatment on the stemness of neural progenitor cells at 2 days post - treatment were analyzed by Western blotting using an anti - SOX2 antibody ( n = 3 biological replicates ) .

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B Effects of CRH treatment on the stemness of neural progenitor cells at 2 days post-treatment were analyzed by Western blotting using an anti-SOX2 antibody (n = 3 biological replicates).


Effects of CRHR1 overexpression on the ( C ) neurosphere

Effects of CRHR1 overexpression on the (C) neurosphere formation


Effects of CRHR1 overexpression on the ( D ) SOX2 expression ( n = 3 biological replicates ) .

Effects of CRHR1 overexpression on the (D) SOX2 expression (n = 3 biological replicates).


E CRHR1 knockdown efficiency of shRNAs used in this study ( n = 4 biological replicates ) .

E CRHR1 knockdown efficiency of shRNAs used in this study (n = 4 biological replicates).


Effects of CRHR1 knockdown on the ( F ) neurosphere formation ,

Effects of CRHR1 knockdown on the (F) neurosphere formation,


Effects of CRHR1 knockdown on the ( G ) SOX2 expression n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

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Effects of CRHR1 knockdown on the (G) SOX2 expression n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).


Effects of CRHR1 knockdown on the ( H ) neurosphere morphology , n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

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Effects of CRHR1 knockdown on the (H) neurosphere morphology, n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).


Effects of CRHR1 knockdown on the ( I ) βIII - tubulin expression . n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

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Effects of CRHR1 knockdown on the (I) βIII-tubulin expression. n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).


J , K Immunostaining using anti - GFP ( green ) together with ( J ) anti - βIII - tubulin ( clone TuJ1 ; red ) ( n = 5 biological replicates ) or ( K ) anti - GFAP ( red ) antibodies ( n = 3 biological replicates ) after in vitro differentiation of E14 . 5 neural progenitor cells transduced with a lentiviral vector expressing shCRHR1 .

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J, K Immunostaining using anti-GFP (green) together with (J) anti-βIII-tubulin (clone TuJ1; red) (n = 5 biological replicates) or (K) anti-GFAP (red) antibodies (n = 3 biological replicates) after in vitro differentiation of E14.5 neural progenitor cells transduced with a lentiviral vector expressing shCRHR1.


A Embryonic brains that were intraventricularly electroporated CRHR1 - expressing plasmids at E13 . 5 were harvested at E15 . 5 and immunostained using an anti - GFP ( green ) and SOX2 ( red ) antibodies . B Quantification of the cell fraction in each laminar layer in ( A ) ( n = 3 biological replicates ) . C Percentage of SOX2 + cells among GFP + cells in ( A ) ( n = 4 biological replicates ) .

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A Embryonic brains that were intraventricularly electroporated CRHR1-expressing plasmids at E13.5 were harvested at E15.5 and immunostained using an anti-GFP (green) and SOX2 (red) antibodies. B Quantification of the cell fraction in each laminar layer in (A) (n = 3 biological replicates). C Percentage of SOX2+ cells among GFP+ cells in (A) (n = 4 biological replicates).


D Embryonic brains were electroporated with shCRHR1 plasmids with or without shCRHR1 - resistant CRHR1 expression vectors at E14 . 5 , and samples were harvested at E17 . 5 for anti - GFP immunolabeling . GFP + cell localization was quantified ( bottom panel ) ( n = 5 biological replicates ) .

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D Embryonic brains were electroporated with shCRHR1 plasmids with or without shCRHR1-resistant CRHR1 expression vectors at E14.5, and samples were harvested at E17.5 for anti-GFP immunolabeling. GFP+ cell localization was quantified (bottom panel) (n = 5 biological replicates).


E , F Brains electroporated with CRHR1 expression vectors at E13 . 5 were harvested at E15 . 5 and co - immunostained using anti - GFP ( green ) together with ( E ) anti - TBR2 ( red ) or ( F ) anti - CTIP2 ( red ) antibodies . n = 3 biological replicates .

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E, F Brains electroporated with CRHR1 expression vectors at E13.5 were harvested at E15.5 and co-immunostained using anti-GFP (green) together with (E) anti-TBR2 (red) or (F) anti-CTIP2 (red) antibodies. n = 3 biological replicates.


G E14 . 5 Embryonic brains that were electroporated with CRHR1 - expression vectors at E13 . 5 were pulse labeled with EdU and harvested at E15 . 5 . Samples were then visualized for GFP ( green ) , EdU ( blue ) and Ki67 ( red ) ( n = 4 biological replicates ) .

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G E14.5 Embryonic brains that were electroporated with CRHR1-expression vectors at E13.5 were pulse labeled with EdU and harvested at E15.5. Samples were then visualized for GFP (green), EdU (blue) and Ki67 (red) (n = 4 biological replicates).


Analysis of the effects of CRHR1 knockdown on neural stemness - and cell - fate - regulating transcription factors in neural progenitor cells by ( A ) qPCR at 2 days post - transduction .

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Analysis of the effects of CRHR1 knockdown on neural stemness- and cell-fate-regulating transcription factors in neural progenitor cells by (A) qPCR at 2 days post-transduction.


Analysis of the effects of CRHR1 knockdown on neural stemness - and cell - fate - regulating transcription factors in neural progenitor cells by ( B ) Western blotting ( n = 3 biological replicates ) at 2 days post - transduction .

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Analysis of the effects of CRHR1 knockdown on neural stemness- and cell-fate-regulating transcription factors in neural progenitor cells by (B) Western blotting (n = 3 biological replicates) at 2 days post-transduction.


The effects of CRHR1 overexpression on REST expression in neural progenitor cells analyzed by qPCR


The effects of CRHR1 overexpression on REST expression in neural progenitor cells analyzed by ( B ) Western blotting ( n = 6 biological replicates ) .

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The effects of CRHR1 overexpression on REST expression in neural progenitor cells analyzed by (B) Western blotting (n = 6 biological replicates).


E Double - immunolabeling of E15 . 5 brain sections electroporated in utero with CRHR1 - expressing plasmid with or without shREST vectors at E13 . 5 using anti - GFP ( green ) and SOX2 ( red ) antibodies .

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E Double-immunolabeling of E15.5 brain sections electroporated in utero with CRHR1-expressing plasmid with or without shREST vectors at E13.5 using anti-GFP (green) and SOX2 (red) antibodies.


F , G Quantification of ( F ) GFP + cell distribution ( n ≥ 4 biological replicates ) and ( G ) percentage of SOX2 + cells ( n ≥ 3 biological replicates ) in ( E ) .

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F, G Quantification of (F) GFP+ cell distribution (n ≥ 4 biological replicates) and (G) percentage of SOX2+ cells (n ≥ 3 biological replicates) in (E).


H Embryonic brains were electroporated with shCRHR1 plasmids with or without REST expression vectors at E14 . 5 , and brain samples were harvested at E17 . 5 for anti - GFP immunolabeling . I Quantification of GFP + cell localization in ( H )

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H Embryonic brains were electroporated with shCRHR1 plasmids with or without REST expression vectors at E14.5, and brain samples were harvested at E17.5 for anti-GFP immunolabeling. I Quantification of GFP+ cell localization in (H)


A , B Western blot analysis of E14 . 5 neural progenitor cells transduced with a lentiviral vector expressing ( A ) CRHR1 or ( B ) shCRHR1 using an anti - CREB and anti - phospho - CREB ( pCREB ) ( S133 ) antibodies . n = 5 biological replicates .

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A, B Western blot analysis of E14.5 neural progenitor cells transduced with a lentiviral vector expressing (A) CRHR1 or (B) shCRHR1 using an anti-CREB and anti-phospho-CREB (pCREB) (S133) antibodies. n = 5 biological replicates.


C E14 . 5 neural progenitor cells were transduced with CRHR1 - containing lentiviral vectors . At 2 days post - transduction , cells were treated with 10 µM H89 ( PKA inhibitor ) or 1 µM KN93 ( CaM kinase inhibitor ) for 8 h and harvested for Western blot analysis for CREB ( top panel ) . Band intensities were quantified ( bottom panel ) ( n = 3 biological replicates ) .

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C E14.5 neural progenitor cells were transduced with CRHR1-containing lentiviral vectors. At 2 days post-transduction, cells were treated with 10 µM H89 (PKA inhibitor) or 1 µM KN93 (CaM kinase inhibitor) for 8 h and harvested for Western blot analysis for CREB (top panel). Band intensities were quantified (bottom panel) (n = 3 biological replicates).


D Double - immunolabeling of E15 . 5 brain sections electroporated in utero with CRHR1 - expressing plasmid at E13 . 5 using anti - GFP ( green ) and pCREB ( S133 ) ( red ) as primary antibodies . Immunostaining was quantified ( right panel ) ( n = 5 biological replicates ) . White inset boxes were magnified and shown in the panels D ' and D ' ' .

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D Double-immunolabeling of E15.5 brain sections electroporated in utero with CRHR1-expressing plasmid at E13.5 using anti-GFP (green) and pCREB (S133) (red) as primary antibodies. Immunostaining was quantified (right panel) (n = 5 biological replicates). White inset boxes were magnified and shown in the panels D' and D''.


E Double - immunolabeling of E15 . 5 brain sections electroporated in utero with CRHR1 - expressing plasmid with or without a vector expressing CREB S133A , a dominant negative mutant form of CREB , at E13 . 5 using anti - GFP ( green ) and SOX2 ( red ) antibodies .

E Double-immunolabeling of E15.5 brain sections electroporated in utero with CRHR1-expressing plasmid with or without a vector expressing CREB S133A, a dominant negative mutant form of CREB, at E13.5 using anti-GFP (green) and SOX2 (red) antibodies.


F , G Quantification of ( F ) GFP + cell distribution ( n = 4 biological replicates ) and ( G ) percentage of SOX2 + cells ( n ≥ 3 biological replicates ) in ( E ) .

F, G Quantification of (F) GFP+ cell distribution (n = 4 biological replicates) and (G) percentage of SOX2+ cells (n ≥ 3 biological replicates) in (E).


H Embryonic brains were electroporated with shCRHR1 plasmids with or without CREB expression vectors at E14 . 5 , and then brain samples were harvested at E17 . 5 for anti - GFP immunolabeling . I Quantification of GFP + cell localization in ( L ) ( n ≥ 4 biological replicates ) .

H Embryonic brains were electroporated with shCRHR1 plasmids with or without CREB expression vectors at E14.5, and then brain samples were harvested at E17.5 for anti-GFP immunolabeling. I Quantification of GFP+ cell localization in (L) (n ≥ 4 biological replicates).


( A ) qPCR of E14 . 5 neural progenitor cells transduced with CREB - expressing lentiviral vectors , measuring REST

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(A) qPCR of E14.5 neural progenitor cells transduced with CREB-expressing lentiviral vectors, measuring REST mRNA


( B ) Western blot analyses ( n = 3 biological replicates ) of E14 . 5 neural progenitor cells transduced with CREB - expressing lentiviral vectors , measuring REST protein levels , respectively .

(B) Western blot analyses (n = 3 biological replicates) of E14.5 neural progenitor cells transduced with CREB-expressing lentiviral vectors, measuring REST protein levels, respectively.


Dose - dependent inhibition of endogenous REST expression by a CREB inhibitor , 666 - 15 . E14 . 5 neural progenitor cells were treated with the indicated concentrations of 666 - 15 for 48 h and subjected to ( C )

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Dose-dependent inhibition of endogenous REST expression by a CREB inhibitor, 666-15. E14.5 neural progenitor cells were treated with the indicated concentrations of 666-15 for 48 h and subjected to (C) qPCR


Dose - dependent inhibition of endogenous REST expression by a CREB inhibitor , 666 - 15 . E14 . 5 neural progenitor cells were treated with the indicated concentrations of 666 - 15 for 48 h and subjected to ( D ) Western blot

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Dose-dependent inhibition of endogenous REST expression by a CREB inhibitor, 666-15. E14.5 neural progenitor cells were treated with the indicated concentrations of 666-15 for 48 h and subjected to (D) Western blot analyses


Inhibition of CRHR1 - induced REST expression by 666 - 15 . E14 . 5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666 - 15 , and after 48 h , cells were lysed and subjected to ( E )

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Inhibition of CRHR1-induced REST expression by 666-15. E14.5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666-15, and after 48 h, cells were lysed and subjected to (E) qPCR


Inhibition of CRHR1 - induced REST expression by 666 - 15 . E14 . 5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666 - 15 , and after 48 h , cells were lysed and subjected to ( F ) Western blot analyses ( n = 4 biological replicates ) .

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Inhibition of CRHR1-induced REST expression by 666-15. E14.5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666-15, and after 48 h, cells were lysed and subjected to (F) Western blot analyses (n = 4 biological replicates).


H Luciferase activity was measured using E14 . 5 neural progenitor cells transfected with CREB expression vectors and pGL3 - Basic luciferase plasmids harboring the wild - type or mutated REST promoter at 2 days post - transfection ( n = 3 biological replicates ) .

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H Luciferase activity was measured using E14.5 neural progenitor cells transfected with CREB expression vectors and pGL3-Basic luciferase plasmids harboring the wild-type or mutated REST promoter at 2 days post-transfection (n = 3 biological replicates).


I A representative gel image of ChIP analysis showing association of CREB proteins with regions of the REST promoter by PCR amplification of putative CRE sequences in the REST promoter ( n = 3 biological replicates ) .

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I A representative gel image of ChIP analysis showing association of CREB proteins with regions of the REST promoter by PCR amplification of putative CRE sequences in the REST promoter (n = 3 biological replicates).


J Immunolabeling of E15 . 5 brain sections electroporated in utero with CREB - expressing plasmids with or without shREST vectors at E13 . 5 using anti - GFP antibody . GFP + cell localization was quantified ( right panel ) ( n ≥ 5 biological replicates ) . The upper and lower dashed lines indicate the boundaries between the CP and IZ , and IZ and VZ / SVZ , respectively .

J Immunolabeling of E15.5 brain sections electroporated in utero with CREB-expressing plasmids with or without shREST vectors at E13.5 using anti-GFP antibody. GFP+ cell localization was quantified (right panel) (n ≥ 5 biological replicates). The upper and lower dashed lines indicate the boundaries between the CP and IZ, and IZ and VZ/SVZ, respectively.


3 ‐ month ‐ old wild ‐ type ( C57BL / 6 ) , Ltbr − / − , Lta − / − , LIGHT − / − , and Ltbr − / − Hox11 − / − mice ; percentage of mice with at least one ANA .

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3‐month‐old wild‐type (C57BL/6), Ltbr−/−, Lta−/−, LIGHT−/−, and Ltbr−/−Hox11−/− mice; percentage of mice with at least one ANA.


6 ‐ month ‐ old Ltbr − / − mice ( n = 15 ) ; titers of individual mice .

6‐month‐old Ltbr−/− mice (n = 15); titers of individual mice.


LIA of 6 ‐ month ‐ old Ltbr − / − mice , wild ‐ type mice , and 3 ‐ month ‐ old Rorγt ‐ Ltb − / − mice .

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LIA of 6‐month‐old Ltbr−/− mice, wild‐type mice, and 3‐month‐old Rorγt‐Ltb−/− mice.


LTβR ‐ Fc or control immunoglobulin ( Ig ) ‐ treated wild ‐ type mice ; percentage of mice with at least one ANA . E denotes gestational day . * ANA were determined with LIA at the age of 3 months . * * ANA were determined with LIA 3 months after the start of treatment .

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LTβR‐Fc or control immunoglobulin (Ig)‐treated wild‐type mice; percentage of mice with at least one ANA. E denotes gestational day. *ANA were determined with LIA at the age of 3 months. **ANA were determined with LIA 3 months after the start of treatment.


3 ‐ month ‐ old cell ‐ specific LT knockout mice ; percentage of mice with at least one ANA .

3‐month‐old cell‐specific LT knockout mice; percentage of mice with at least one ANA.


Bone marrow cells from wild ‐ type or Ltbr − / − mice were transferred at neonatal age into lethally irradiated wild ‐ type or Ltbr − / − mice ; percentage of mice with at least one ANA . ANA were determined with LIA at the age of 3 months .

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Bone marrow cells from wild‐type or Ltbr−/− mice were transferred at neonatal age into lethally irradiated wild‐type or Ltbr−/− mice; percentage of mice with at least one ANA. ANA were determined with LIA at the age of 3 months.


Thymic reconstitution was analyzed by counting total cell number and flow cytometry for cell percentages . Numbers represent the percentages within the indicated regions ( left panel ) . No significant differences were found . Data represent means ± s . e . m .

Thymic reconstitution was analyzed by counting total cell number and flow cytometry for cell percentages. Numbers represent the percentages within the indicated regions (left panel). No significant differences were found. Data represent means ± s.e.m.


T ‐ cell reconstitution of spleen was assessed by flow cytometry . Numbers represent the percentages within the indicated regions .

T‐cell reconstitution of spleen was assessed by flow cytometry. Numbers represent the percentages within the indicated regions.


Total IgG from nude mice engrafted with Ltbr − / − or wild ‐ type thymi was determined by ELISA . No significant differences were found . Data represent means ± s . e . m .

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Total IgG from nude mice engrafted with Ltbr−/− or wild‐type thymi was determined by ELISA. No significant differences were found. Data represent means ± s.e.m.


Sera were collected 3 months after transplantation and tested for ANA ; percentage of mice with at least one ANA reactivity .

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Sera were collected 3 months after transplantation and tested for ANA; percentage of mice with at least one ANA reactivity.


Ltbr − / − mice were treated or not with antibiotics from birth ( upper panel ) , conventionalized or germfree wild ‐ type mice were treated with the LTβR ‐ Fc fusion protein from gestational day 18 until 6 weeks after birth ( lower panel ) ; percentage of mice with at least one ANA . ANA were tested with LIA at the age of 3 months .

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Ltbr−/− mice were treated or not with antibiotics from birth (upper panel), conventionalized or germfree wild‐type mice were treated with the LTβR‐Fc fusion protein from gestational day 18 until 6 weeks after birth (lower panel); percentage of mice with at least one ANA. ANA were tested with LIA at the age of 3 months.


Real ‐ time PCR for SFB in luminal , mucosal , and fecal samples of multiple ANA ‐ positive and ANA ‐ negative Ltbr − / − mice . Analysis with ANOVA , P = 0 . 02 for differences between groups , n = 4 mice per group . Data represent means ± s . e . m .

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Real‐time PCR for SFB in luminal, mucosal, and fecal samples of multiple ANA‐positive and ANA‐negative Ltbr−/− mice. Analysis with ANOVA, P = 0.02 for differences between groups, n = 4 mice per group. Data represent means ± s.e.m.


C3H / HeN germfree pregnant mice were treated with the LTβR ‐ Fc fusion protein on day 16 and 18 of gestation to prevent the development of PP in their offspring . Pups were also treated in the neonatal period to prevent the development of ILF . Mice were colonized at 8 weeks of age with SFB or E . coli and were sacrificed on d20 or on d60 postcolonization ; percentage of mice with at least one ANA . E denotes gestational day .

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C3H/HeN germfree pregnant mice were treated with the LTβR‐Fc fusion protein on day 16 and 18 of gestation to prevent the development of PP in their offspring. Pups were also treated in the neonatal period to prevent the development of ILF. Mice were colonized at 8 weeks of age with SFB or E. coli and were sacrificed on d20 or on d60 postcolonization; percentage of mice with at least one ANA. E denotes gestational day.


LTβR ‐ Fc ‐ treated wild ‐ type or IL17R − / − mice ; percentage of mice with at least one ANA . E denotes gestational day . ANA were determined with LIA at the age of 3

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LTβR‐Fc‐treated wild‐type or IL17R−/− mice; percentage of mice with at least one ANA. E denotes gestational day. ANA were determined with LIA at the age of 3 months


( A ) Time course of rapid internalization of fluorescent α - synuclein fibrils by CADcells ( for characterization of α - synuclein assemblies , see Figure EV 1A ) . Internalization was measured by recording ATTO - 550 - positive neuron - like cells by flow cytometry . Percentage of ATTO - 550 - positive cells was quantified ( mean ± s . e . m ) ( left panel ) and representative histograms of ATTO - 550 - positive cells are showed on the right panel ( a . u . arbitrary units ) . Similarly , α - synuclein fibrils internalization was also confirmed by fluorescent microscopy ( see Figure EV 1B ) .

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(A) Time course of rapid internalization of fluorescent α-synuclein fibrils by CADcells (for characterization of α-synuclein assemblies, see Figure EV 1A). Internalization was measured by recording ATTO-550-positive neuron-like cells by flow cytometry. Percentage of ATTO-550-positive cells was quantified (mean ±s.e.m) (left panel) and representative histograms of ATTO-550-positive cells are showed on the right panel (a.u. arbitrary units). Similarly, α-synuclein fibrils internalization was also confirmed by fluorescent microscopy (see Figure EV 1B).


B ) Representative images of donor ( upper panel ) and acceptor cells ( lower panel ) after 24 h co - culture . Donor cells were loaded with α - synuclein fibrils prior to co - culture with GFP - transfected acceptor cells ; in red : α - synuclein fibrils , in green : acceptor cells and in blue : nuclei . Scale bar represents 10 μm . ( n = 3 independent experiments ) . A larger field where donor and acceptor cells are shown is presented in Figure EV 3A . ( C ) Percentage of donor and acceptor cells containing α - synuclein fibrils after co - culture as in B : all acceptor cells received α - synuclein fibrils . ( D ) Quantification of the number of α - synuclein fibrils in donor and acceptor cells after co - culture as in B . Donor cells contain around 70 α - synuclein fibrils puncta ( median ) while acceptor cells contain 38 α - synuclein fibrils puncta , respectively ( * * * * , p < 0 . 0001 by two - tailed Mann Whitney test ) . ( E ) Quantification of the average size of α - synuclein fibrillar foci in donor and acceptor cells after co - culture as in B . ( * * * * , p < 0 . 0001 by two - tailed Mann Whitney test ) . In Figure EV 3 is shown an example of α - synuclein fibrillar puncta detection in an acceptor cell . After detection , the number and the size of foci were determined using the ICY software .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of donor (upper panel) and acceptor cells (lower panel) after 24 h co-culture. Donor cells were loaded with α-synuclein fibrils prior to co-culture with GFP-transfected acceptor cells; in red: α-synuclein fibrils, in green: acceptor cells and in blue: nuclei. Scale bar represents 10 μm. (n = 3 independent experiments). A larger field where donor and acceptor cells are shown is presented in Figure EV 3A.(C) Percentage of donor and acceptor cells containing α-synuclein fibrils after co-culture as in B: all acceptor cells received α-synuclein fibrils.(D) Quantification of the number of α-synuclein fibrils in donor and acceptor cells after co-culture as in B. Donor cells contain around 70 α-synuclein fibrils puncta (median) while acceptor cells contain 38 α-synuclein fibrils puncta, respectively (****, p < 0.0001 by two-tailed Mann Whitney test).(E) Quantification of the average size of α-synuclein fibrillar foci in donor and acceptor cells after co-culture as in B. (****, p < 0.0001 by two-tailed Mann Whitney test). In Figure EV 3 is shown an example of α-synuclein fibrillar puncta detection in an acceptor cell. After detection, the number and the size of foci were determined using the ICY software.


( A ) Representative confocal images showing ChFP - α - syn transfected cells ( in red ) cultured alone ( upper ) or co - cultured with donor cells containing fluorescent Alexa - 488 α - synuclein fibrils ( in green ) for 72 h ( bottom ) . The arrow is pointing out representative α - synuclein fibrils Alexa - 488 co - localized with ChFP - α - syn puncta . Scale bar represents 10 μm . Nuclei are stained with DAPI ( blue ) ( n = 3 independent experiments ) . ( B ) Quantification of the number of ChFP - α - synuclein puncta in ChFP - α - syn acceptor cells showed a significant increase in ChFP - α - synuclein puncta number when cells were co - cultured with donor cells containing α - synuclein fibrils ( * * * , p < 0 . 001 by two - tailed Mann Whitney test ) . ( C ) Quantification of the percentage of co - localization of α - synuclein fibrils Alexa - 488 and ChFP - α - syn puncta . Data shows mean ± s . e . m . After co - culture , 11 % of the transferred α - synuclein fibrils Alexa - 488 co - localized with ChFP - α - syn

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal images showing ChFP-α-syn transfected cells (in red) cultured alone (upper) or co-cultured with donor cells containing fluorescent Alexa-488 α-synuclein fibrils (in green) for 72 h (bottom). The arrow is pointing out representative α-synuclein fibrils Alexa-488 co-localized with ChFP-α-syn puncta. Scale bar represents 10 μm. Nuclei are stained with DAPI (blue) (n=3 independent experiments).(B) Quantification of the number of ChFP-α-synuclein puncta in ChFP-α-syn acceptor cells showed a significant increase in ChFP-α-synuclein puncta number when cells were co-cultured with donor cells containing α-synuclein fibrils (***, p < 0.001 by two-tailed Mann Whitney test).(C) Quantification of the percentage of co-localization of α-synuclein fibrils Alexa-488 and ChFP-α-syn puncta. Data shows mean ± s.e.m. After co-culture, 11% of the transferred α-synuclein fibrils Alexa-488 co-localized with ChFP-α-syn puncta


( A ) Representative images of donor cells ( upper panel ) and acceptor cells ( bottom panel ) of the co - culture system explained in Figure EV 2E ; in red : α - synuclein fibrils ATTO - 550 , in green : α - synuclein fibrils Alexa - 488 and in blue : nuclei . Scale bar represents 10 μm ( n = 3 independent experiments ) . In insets , arrows point to co - localized ATTO - 550 and Alexa - 488 α - synuclein fibrils puncta whereas arrowheads point to puncta of α - synuclein Alexa - 488 fibrils that do not co - localize with fibrillar ATTO - 550 α - synuclein .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of donor cells (upper panel) and acceptor cells (bottom panel) of the co-culture system explained in Figure EV 2E; in red: α-synuclein fibrils ATTO-550, in green: α-synuclein fibrils Alexa-488 and in blue: nuclei. Scale bar represents 10 μm (n=3 independent experiments). In insets, arrows point to co-localized ATTO-550 and Alexa-488 α-synuclein fibrils puncta whereas arrowheads point to puncta of α-synuclein Alexa-488 fibrils that do not co-localize with fibrillar ATTO-550 α-synuclein.


( B ) Quantification of the percentage of Alexa - 488 α - synuclein fibrils co - localizing with α - ATTO - 550 α - synuclein fibrils and the reverse co - localization in discrete puncta in donor and acceptor cells as in A . Both measurements revealed high co - localization of the two fluorophores in donor cells ( white bar ) , but less in acceptor cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the percentage of Alexa-488 α-synuclein fibrils co-localizing with α- ATTO-550 α-synuclein fibrils and the reverse co-localization in discrete puncta in donor and acceptor cells as in A. Both measurements revealed high co-localization of the two fluorophores in donor cells (white bar), but less in acceptor cells.


( C ) The amount of fibrils taken up by CAD cells exposed to 1µM fibrils , of fibrils remaining in the cells and exported into the medium after 24h incubation were quantified by a filter trap assay . Data are mean ± s . d ( n = 3 independent measurements , filtered in duplicate ) . The standard fluorescence curve for increasing ATTO550 - α - synucleinfibrils concentrations is given .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The amount of fibrils taken up by CAD cells exposed to 1µM fibrils, of fibrils remaining in the cells and exported into the medium after 24h incubation were quantified by a filter trap assay. Data are mean ± s.d (n=3 independent measurements, filtered in duplicate). The standard fluorescence curve for increasing ATTO550-α-synucleinfibrils concentrations is given.


( D ) Representative images of GFP - transfected acceptor cells that were either ( i ) co - cultured with donor cells ( upper panel , Co - culture ) , ( ii ) cultured with the conditioned medium of donor cells ( middle panel , CM ) or ( iii ) physically separated from donor cells using a filter ( bottom panel , Filter ) . Prior to culture , donor cells were loaded with ATTO - 550 α - synuclein fibrils . In red : α - synuclein fibrils , in green : acceptor cells and in blue : nuclei . Scale bar represents 10 μm ( n = 3 independent experiments ) . ( E ) Quantification of the percentage of acceptor cells containing α - synuclein fibrils from images such as those presented in C . When acceptor cells were cultured with the conditioned medium from donor cells previously treated with α - synucleinfibrils ( not diluted , concentrated or diluted ) or co - cultured with a filter , the percentage of acceptor cells containing α - synuclein puncta was low . Data shows mean ± s . e . m ( * * * , p < 0 . 001 ; by two - tailed Student ' s t - test ) . ( F ) Quantification of the number of puncta of α - synuclein fibrils per acceptor cell from D . While in co - culture condition the number of α - synuclein puncta in acceptor cells is on average 35 ( median ) , this number was on average 1 puncta per acceptor cells in CM and filter conditions , respectively ( * * * , p < 0 . 001 by two - tailed Mann Whitney 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of GFP-transfected acceptor cells that were either (i) co-cultured with donor cells (upper panel, Co-culture), (ii) cultured with the conditioned medium of donor cells (middle panel, CM) or (iii) physically separated from donor cells using a filter (bottom panel, Filter). Prior to culture, donor cells were loaded with ATTO-550 α-synuclein fibrils. In red: α-synuclein fibrils, in green: acceptor cells and in blue: nuclei. Scale bar represents 10 μm (n = 3 independent experiments).(E) Quantification of the percentage of acceptor cells containing α-synuclein fibrils from images such as those presented in C. When acceptor cells were cultured with the conditioned medium from donor cells previously treated with α-synucleinfibrils (not diluted, concentrated or diluted) or co-cultured with a filter, the percentage of acceptor cells containing α-synuclein puncta was low. Data shows mean ± s.e.m (***, p < 0.001; by two-tailed Student's t-test).(F) Quantification of the number of puncta of α-synuclein fibrils per acceptor cell from D. While in co-culture condition the number of α-synuclein puncta in acceptor cells is on average 35 (median), this number was on average 1 puncta per acceptor cells in CM and filter conditions, respectively (***, p < 0.001 by two-tailed Mann Whitney test).


( A ) Representative images of control cells ( upper panel ) or cells treated with α - synucleinfibrilsATTO - 550 ( bottom panel ) for 16 hours ; in red : α - synucleinfibrils and in green : WGAAlexa - 488 ( plasma membrane dye marker ) , blue : DAPI . Scale bar represents 10 μm ( n = 3 independent experiments ) . ( B ) Relative percentage of TNT - connected cells after α - synucleinfibrils treatment as in A shows an increase in TNT number upon fibrils treatment . Data are mean ± s . e . m ( * * * , p < 0 . 001 by paired , two - tailed 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

(A) Representative images of control cells (upper panel) or cells treated with α-synucleinfibrilsATTO-550 (bottom panel) for 16 hours; in red: α-synucleinfibrils and in green: WGAAlexa-488 (plasma membrane dye marker), blue: DAPI. Scale bar represents 10 μm (n = 3 independent experiments).(B) Relative percentage of TNT-connected cells after α-synucleinfibrils treatment as in A shows an increase in TNT number upon fibrils treatment. Data are mean ± s.e.m (***, p < 0.001 by paired, two-tailed Student's t-test).


( C ) Confocal images showing one TNT connecting two cells loaded with α - synucleinfibrilsATTO - 550 ( in red ) 16 hours after co - culture and stained with WGAAlexa - 488 ( in blue ) . The image represents a Z - projection of the several middle stacks where the TNT is located ( top panel ) . Insets show 3D reconstructions of the TNT using ICY software ( middle panel : merge ; bottom panel : red channel ) . Several α - synucleinfibrils ( red ) are present in the lumen of the TNT ( WGAAlexa - 488 in white ) . Scale bars represent 10 μm ( confocal images ) and 4 μm ( insets ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images showing one TNT connecting two cells loaded with α-synucleinfibrilsATTO-550 (in red) 16 hours after co-culture and stained with WGAAlexa-488 (in blue). The image represents a Z-projection of the several middle stacks where the TNT is located (top panel). Insets show 3D reconstructions of the TNT using ICY software (middle panel: merge; bottom panel: red channel). Several α-synucleinfibrils (red) are present in the lumen of the TNT (WGAAlexa-488 in white). Scale bars represent 10 μm (confocal images) and 4 μm (insets).


( D ) Confocal image showing TNT connecting a donor cell containing α - synucleinfibrils and GFP transfected acceptor cell ( top panel ) strongly suggesting α - synucleinfibrils transfer from donor to acceptor cell . Insets show 3D reconstructions of TNT containing α - synucleinfibrils in the lumen of the TNT ; in red : α - synucleinfibrils , in green : GFP transfected acceptor cells in blue : WGAAlexa - 488 . Scale bars represent 10 μm and 3 μm for the insets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal image showing TNT connecting a donor cell containing α-synucleinfibrils and GFP transfected acceptor cell (top panel) strongly suggesting α-synucleinfibrils transfer from donor to acceptor cell. Insets show 3D reconstructions of TNT containing α-synucleinfibrils in the lumen of the TNT; in red: α-synucleinfibrils, in green: GFP transfected acceptor cells in blue: WGAAlexa-488. Scale bars represent 10 μm and 3 μm for the insets.


( E ) Relative percentage of TNTs in cells containing α - synucleinfibrils untransfected ( control ) or transfected with Myosin 10 ( Myosin 10 ) . Overexpression of Myosin 10 in the donor cell population led to an increase in the number of TNTs .

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

(E) Relative percentage of TNTs in cells containing α-synucleinfibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Overexpression of Myosin 10 in the donor cell population led to an increase in the number of TNTs.


( F ) Quantification of the average number of ATTO - 550α - synucleinfibrils per acceptor cell after 24 hours co - culture with donor cells containing α - synucleinfibrils untransfected ( control ) or transfected with Myosin 10 ( Myosin 10 ) . Note that Myosin 10 overexpression increases both the transfer of DiIvesicles and the number of transferred α - synuclein puncta in acceptor cells ; n = 3 independent experiments ( * * , p < 0 . 01 by two - tailed Mann Whitney 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

(F) Quantification of the average number of ATTO-550α-synucleinfibrils per acceptor cell after 24 hours co-culture with donor cells containing α-synucleinfibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Note that Myosin 10 overexpression increases both the transfer of DiIvesicles and the number of transferred α-synuclein puncta in acceptor cells; n = 3 independent experiments (**, p < 0.01 by two-tailed Mann Whitney test).


( G ) Quantification of the relative proportion of TNTs in co - culture of donor cells containing α - synuclein puncta and GFP acceptor cells under sub - confluent or sparse culture conditions . Data are mean ± s . e . m ; n = 3 independent experiments ( * , p < 0 . 05 by paired , two - tailed Student ' s t - test ) . ( H ) Quantification of the percentage of acceptor cells containing α - synuclein puncta of the co - culture in G . n = 3 independent experiments ( * , p < 0 . 05 by two - tailed Mann Whitney test ) . Condition impairing TNT formation ( i . e . , sparse condition ) induces a drastic decrease of TNT number and α - synucleinfibrils transfer compared to condition allowing TNT formation ( sub - confluent condition ) .

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

(G) Quantification of the relative proportion of TNTs in co-culture of donor cells containing α-synuclein puncta and GFP acceptor cells under sub-confluent or sparse culture conditions. Data are mean ± s.e.m; n = 3 independent experiments (*, p < 0.05 by paired, two-tailed Student's t-test).(H) Quantification of the percentage of acceptor cells containing α-synuclein puncta of the co-culture in G. n = 3 independent experiments (*, p < 0.05 by two-tailed Mann Whitney test). Condition impairing TNT formation (i.e., sparse condition) induces a drastic decrease of TNT number and α-synucleinfibrils transfer compared to condition allowing TNT formation (sub-confluent condition).


( A ) Subcellular localization of α - synucleinfibrils in donor cells after 24 hours co - culture . The left panels show representative images of co - localization of α - synuclein puncta with early endosomes ( EEA1 , top panel ) , recycling endosomes ( Vamp3 , middle panel ) and lysosomal vesicles ( Lamp1 , bottom panel ) ( see arrows for co - localized spots ) ; in red : α - synucleinfibrils , in green : EEA1 , Vamp3 or Lamp1 and in blue : nuclei . Scale bar represents 10 μm . On the right , the bar graph represents the percentage of α - synucleinpuncta co - localized with endo - lysosomal vesicles in A . Data are mean ± s . e . m . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Subcellular localization of α-synucleinfibrils in donor cells after 24 hours co-culture. The left panels show representative images of co-localization of α-synuclein puncta with early endosomes (EEA1, top panel), recycling endosomes (Vamp3, middle panel) and lysosomal vesicles (Lamp1, bottom panel) (see arrows for co-localized spots); in red: α-synucleinfibrils, in green: EEA1, Vamp3 or Lamp1 and in blue: nuclei. Scale bar represents 10 μm. On the right, the bar graph represents the percentage of α-synucleinpuncta co-localized with endo-lysosomal vesicles in A. Data are mean ± s.e.m. from three independent experiments.


( B ) Confocal images showing a TNT connecting two cells loaded with α - synucleinfibrilsATTO - 550 immunostained with Lamp1 ( in green ) . Labelling of TNTs is performed using a plasma membrane dye ( WGAAlexa - 488 , in blue ) . The image represents a Z - projection of the several middle stacks where the TNT is located . The merged image with WGA - Alexa 633 ( blue ) , the orthogonal view and insets showing 3D reconstructions of the TNT ( with WGA in white ) are in the right panel . Several α - synuclein fibrillar foci ( in red ) are present in the lumen of the TNT and embedded in Lamp1 positive vesicles ( in green ) . Of note , only tubular structures physically connecting remote cells and not touching the substratum were identified as TNTs to distinguish them from filopodia . Scale bars represent 10 μm ( confocal images ) and 3 μm ( insets ) . The merge of α - synucleinfibrils ( red ) and Lamp1 ( green ) channels and the respective single channels are showed on the left panel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images showing a TNT connecting two cells loaded with α-synucleinfibrilsATTO-550 immunostained with Lamp1 (in green). Labelling of TNTs is performed using a plasma membrane dye (WGAAlexa-488, in blue). The image represents a Z-projection of the several middle stacks where the TNT is located. The merged image with WGA-Alexa 633 (blue), the orthogonal view and insets showing 3D reconstructions of the TNT (with WGA in white) are in the right panel. Several α-synuclein fibrillar foci (in red) are present in the lumen of the TNT and embedded in Lamp1 positive vesicles (in green). Of note, only tubular structures physically connecting remote cells and not touching the substratum were identified as TNTs to distinguish them from filopodia. Scale bars represent 10 μm (confocal images) and 3 μm (insets). The merge of α-synucleinfibrils (red) and Lamp1 (green) channels and the respective single channels are showed on the left panel.


( C ) The top panels show representative images of GFP - transfected acceptor cells after 24 h co - culture with donor cells containing α - synucleinfibrilsATTO - 550 . Immunofluorescence was performed with endo - lysosomal organelle markers ( top panel : EEA1 , middle panel : Vamp3 and Lamp1 : bottom panel ) and revealed that α - synucleinfibrils co - localized with lysosomal vesicles ( see arrow ) ; in red : α - synucleinfibrils , in green : organelle markers and in blue : nuclei . Scale bar represents 10 μm , arrows pinpoint assemblies co - localizing with organelle markers . The bar graph at the bottom shows the quantification of the percentage of co - localization of α - synucleinfibrils with organelle markers , revealing that α - synuclein puncta co - localized mostly with lysosomal vesicles . Data show mean ± s . e . m . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The top panels show representative images of GFP-transfected acceptor cells after 24 h co-culture with donor cells containing α-synucleinfibrilsATTO-550. Immunofluorescence was performed with endo-lysosomal organelle markers (top panel: EEA1, middle panel: Vamp3 and Lamp1: bottom panel) and revealed that α-synucleinfibrils co-localized with lysosomal vesicles (see arrow); in red: α-synucleinfibrils, in green: organelle markers and in blue: nuclei. Scale bar represents 10 μm, arrows pinpoint assemblies co-localizing with organelle markers. The bar graph at the bottom shows the quantification of the percentage of co-localization of α-synucleinfibrils with organelle markers, revealing that α-synuclein puncta co-localized mostly with lysosomal vesicles. Data show mean ± s.e.m. from three independent experiments.


( D ) Representative confocal pictures of control cells loaded with ATTO - 550α - synucleinfibrils and LysoTracker Deep Red ( top panel ) , and of donor ( middle panel ) and acceptor GFP - transfected acceptor cells ( bottom panel , GFP - vector not shown ) cells , loaded with α - synucleinfibrilsATTO - 550 and LysoTracker Deep Red ( middle panel ) and co - cultured for 24 hours ; in red : α - synucleinfibrils , in green : LysoTracker Deep Red and in blue : nuclei . Scale bar represents 10 μm , arrows in inset pinpoint fibrils co - localizing with LysoTracker positive vesicles . ( E ) The percentage of co - localization of LysoTracker positive vesicles with α - synucleinfibrils revealed that 80 % of lysosomal vesicles that transferred from donor to acceptor cells contained α - synucleinfibrils thus demonstrating direct transfer of α - synucleinfibrils from donor to acceptor cells in majority inside lysosomal vesicles derived from donor cells . Data show mean ± s . e . m . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal pictures of control cells loaded with ATTO-550α-synucleinfibrils and LysoTracker Deep Red (top panel), and of donor (middle panel) and acceptor GFP-transfected acceptor cells (bottom panel, GFP-vector not shown) cells, loaded with α-synucleinfibrilsATTO-550 and LysoTracker Deep Red (middle panel) and co-cultured for 24 hours; in red: α-synucleinfibrils, in green: LysoTracker Deep Red and in blue: nuclei. Scale bar represents 10 μm, arrows in inset pinpoint fibrils co-localizing with LysoTracker positive vesicles.(E) The percentage of co-localization of LysoTracker positive vesicles with α-synucleinfibrils revealed that 80% of lysosomal vesicles that transferred from donor to acceptor cells contained α-synucleinfibrils thus demonstrating direct transfer of α-synucleinfibrils from donor to acceptor cells in majority inside lysosomal vesicles derived from donor cells. Data show mean ± s.e.m. from three independent experiments.


( A ) Representative images of control ( upper panel ) and α - synuclein loaded neurons ( middle , 0 . 5µM , and bottom , 1µM , panels ) after 16 h . In red : α - synuclein fibrils , in green : MAP - 2 and in blue : nuclei . Scale bar represents 10 μm .

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(A) Representative images of control (upper panel) and α-synuclein loaded neurons (middle, 0.5µM, and bottom, 1µM, panels) after 16 h. In red: α-synuclein fibrils, in green: MAP-2 and in blue: nuclei. Scale bar represents 10 μm.


( B ) Representative images of donor ( upper panels ) and acceptor neurons ( bottom panels ) after 72 hours in co - culture . Donor neurons were loaded with α - synuclein fibrils prior to co - culture with CTG - labelled acceptor neurons and then cells were labelled either with MAP - 2 ( first and third panels ) or Lamp1 ( second and fourth panels ) . In red : α - synuclein fibrils , in green : CTG , in white : MAP - 2 / Lamp1 and in blue : nuclei . Scale bar represents 5 μm . ( C ) The graph bar shows the percentage of donor ( white bar ) and acceptor ( black bar ) neurons containing α - synuclein puncta . Data show mean ± s . e . m . ( D ) The box plot depicts the number of puncta in donor ( white ) and acceptor ( grey ) neurons . ( E ) Box plot showing the distribution of the average size of α - synuclein puncta in donor ( white ) and acceptor ( grey ) neurons . ( F ) The graph bar represents the percentage of α - synuclein puncta co - localized with lysosomes in donor ( white ) and acceptor ( black ) neurons . Data show mean ± s . e . m . Graphs in C , D , E and F correspond to data from 3 independent experiments analysed after 72 hours of co - culture ( * * * * , p < 0 . 0001 compared to donor by two - tailed Mann Whitney test ) .

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(B) Representative images of donor (upper panels) and acceptor neurons (bottom panels) after 72 hours in co-culture. Donor neurons were loaded with α-synuclein fibrils prior to co-culture with CTG-labelled acceptor neurons and then cells were labelled either with MAP-2 (first and third panels) or Lamp1 (second and fourth panels). In red: α-synuclein fibrils, in green: CTG, in white: MAP-2/ Lamp1 and in blue: nuclei. Scale bar represents 5 μm.(C) The graph bar shows the percentage of donor (white bar) and acceptor (black bar) neurons containing α-synuclein puncta. Data show mean ± s.e.m.(D) The box plot depicts the number of puncta in donor (white) and acceptor (grey) neurons.(E) Box plot showing the distribution of the average size of α-synuclein puncta in donor (white) and acceptor (grey) neurons.(F) The graph bar represents the percentage of α-synuclein puncta co-localized with lysosomes in donor (white) and acceptor (black) neurons. Data show mean ± s.e.m. Graphs in C, D, E and F correspond to data from 3 independent experiments analysed after 72 hours of co-culture (****, p < 0.0001 compared to donor by two-tailed Mann Whitney test).


( B ) Representative images of acceptor neurons that were either ( i ) co - cultured with donor neurons ( upper panel , Co - culture ) , ( ii ) physically separated from donor cells ( middle panel , No contact ) or ( iii ) cultured with the conditioned medium of donor neurons ( bottom panel , CM ) . In red : α - synucleinfibrils , in white : acceptor neurons and in blue : nuclei . Scale bar represents 10 μm .

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(B) Representative images of acceptor neurons that were either (i) co-cultured with donor neurons (upper panel, Co-culture), (ii) physically separated from donor cells (middle panel, No contact) or (iii) cultured with the conditioned medium of donor neurons (bottom panel, CM). In red: α-synucleinfibrils, in white: acceptor neurons and in blue: nuclei. Scale bar represents 10 μm.


( C ) The graph bar shows the percentage of acceptor cells containing α - synuclein puncta from images such as those presented in B . * * * * , p < 0 . 001 compared to the co - culture condition by two - tailed Student ' s t - test . Data show mean ± s . e . m from 3 independent experiments . ( D ) The box plot shows the number of α - synuclein puncta in acceptor neurons in co - culture ( white ) , in CM ( grey ) and in the No contact condition ( light grey ) . * * * * , p < 0 . 001 compared to the co - culture condition by two - tailed Mann Whitney test ( n = 3 independent experiments ) . .

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(C) The graph bar shows the percentage of acceptor cells containing α-synuclein puncta from images such as those presented in B. ****, p < 0.001 compared to the co-culture condition by two-tailed Student's t-test. Data show mean ± s.e.m from 3 independent experiments.(D) The box plot shows the number of α-synuclein puncta in acceptor neurons in co-culture (white), in CM (grey) and in the No contact condition (light grey). ****, p < 0.001 compared to the co-culture condition by two-tailed Mann Whitney test (n=3 independent experiments)..


( E ) Western blot analysis of α - synuclein indicates the presence of some α - synuclein in the CM of neurons loaded with the fibrils , after 72h compared to control medium and to lysate of cells directly exposed to the fibrils . α - tubulin was used as loading control .

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(E) Western blot analysis of α-synuclein indicates the presence of some α-synuclein in the CM of neurons loaded with the fibrils, after 72h compared to control medium and to lysate of cells directly exposed to the fibrils. α-tubulin was used as loading control.


( A ) Representative images of donor ( upper panel ) and acceptor cells ( bottom panel ) after 24 hours in co - culture . Donor neurons were loaded with α - synucleinfibrils prior to co - culture with H2B - GFP - acceptor CAD cells . In red : α - synuclein puncta , in green : acceptor CADs , in white donor neurons and in blue : nuclei . The images of donor cells are representative Z - stack projections of the lower slices where these cells are located , and the images corresponding to acceptor CADs are Z - stack projections of the upper slices , covering the whole cell body . Scale bar represents 10 μm . ( B ) Box plots showing the number ( left side ) and average size ( right side ) of α - synuclein puncta in acceptor CAD cells after 24 hours in co - culture with donor neurons . ( n = 3 independent experiments ) .

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(A) Representative images of donor (upper panel) and acceptor cells (bottom panel) after 24 hours in co-culture. Donor neurons were loaded with α-synucleinfibrils prior to co-culture with H2B-GFP-acceptor CAD cells. In red: α-synuclein puncta, in green: acceptor CADs, in white donor neurons and in blue: nuclei. The images of donor cells are representative Z-stack projections of the lower slices where these cells are located, and the images corresponding to acceptor CADs are Z-stack projections of the upper slices, covering the whole cell body. Scale bar represents 10 μm.(B) Box plots showing the number (left side) and average size (right side) of α-synuclein puncta in acceptor CAD cells after 24 hours in co-culture with donor neurons. (n=3 independent experiments).


( C ) Representative images showing a TNT detected between CAD cells and neurons after 24 hours in co - culture . In green : MAP - 2 , in white : WGA , in red : α - synuclein puncta and in blue nuclei . The merge image in the bottom panel shows in detail the TNT connecting the cells , and the insets and the 3D reconstruction of the neuron ( green ) and the CAD cell ( white ) show the presence of α - synuclein puncta inside the TNT .

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(C) Representative images showing a TNT detected between CAD cells and neurons after 24 hours in co-culture. In green: MAP-2, in white: WGA, in red: α-synuclein puncta and in blue nuclei. The merge image in the bottom panel shows in detail the TNT connecting the cells, and the insets and the 3D reconstruction of the neuron (green) and the CAD cell (white) show the presence of α-synuclein puncta inside the TNT.


( C ) Asynchronous exponentially growing cells were treated with 0 . 2M HU ( + HU ) , then HU was removed from the medium ( - HU ) . SDS - PAGE of total protein extracts taken at the indicated times were used to detect the kinetic of Rad53 phosphorylation upshift ( Rad53 - P = * * ) . Red and green asterisk indicate the time where mec1 - S1991 phosphomutants show altered Rad53 recovery . A 5 - fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC + / - the indicated dose of HU .

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(C) Asynchronous exponentially growing cells were treated with 0.2M HU (+HU), then HU was removed from the medium (-HU). SDS-PAGE of total protein extracts taken at the indicated times were used to detect the kinetic of Rad53 phosphorylation upshift (Rad53-P=**). Red and green asterisk indicate the time where mec1-S1991 phosphomutants show altered Rad53 recovery. A 5-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- the indicated dose of HU.


Analysis of replication fork progression at the single - molecule level by DNA combing . ( C ) Representative images of DNA fibers . Green : ssDNA , red : BrdU . Scale bar corresponds to 20 kb . ( D ) Graph depict the distribution of BrdU track length . WT 90 min ( n = 427 ) and 180 min ( n = 483 ) , mec1 - S1991A 90 min ( n = 309 ) and 180 min ( n = 477 ) .

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Analysis of replication fork progression at the single-molecule level by DNA combing. (C) Representative images of DNA fibers. Green: ssDNA, red: BrdU. Scale bar corresponds to 20 kb. (D) Graph depict the distribution of BrdU track length. WT 90 min (n=427) and 180 min (n=483), mec1-S1991A 90 min (n=309) and 180 min (n=477).


( A ) tRNA level measured by RT - qPCR in asynchronous ( async . ) culture and after 90 min on HU in the indicated strains . Expression is normalized to ACT1 . The repression is expressed as a ratio of HU treated / asynchronous cells in percent .

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(A) tRNA level measured by RT-qPCR in asynchronous (async.) culture and after 90 min on HU in the indicated strains. Expression is normalized to ACT1. The repression is expressed as a ratio of HU treated/asynchronous cells in percent.


( A - D ) Exponentially growing cells were treated with 0 . 2M HU and total protein extracts were collected at the indicated time points ( in min ) , samples were subjected to SDS - PAGE followed by immunoblotting with Rpb1 and Tubulin antibodies . ( B , D ) Quantitation of total Rpb1 over time was done by normalizing Rpb1 levels to tubulin .

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(A-D) Exponentially growing cells were treated with 0.2M HU and total protein extracts were collected at the indicated time points (in min), samples were subjected to SDS-PAGE followed by immunoblotting with Rpb1 and Tubulin antibodies. (B,D) Quantitation of total Rpb1 over time was done by normalizing Rpb1 levels to tubulin.


( E , F ) Drop assay on HU showing a 10 - fold dilution series of cells from exponential SC cultures of the indicated strains that were spotted on SC + / - 200mM HU . ( F ) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution .

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(E,F) Drop assay on HU showing a 10-fold dilution series of cells from exponential SC cultures of the indicated strains that were spotted on SC +/- 200mM HU. (F) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.


( A ) Preparations of chromatin - bound proteins were prepared in triplicate and subjected to SDS - PAGE and immunoblotting with the indicated antibodies in a strain that either does or does not express Rpb3 - TAP Quantification of chromatin - bound Rpb1 - S2P and Rpb1 - S5P is shown on the right . Mcm2 is used as a loading control .

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(A) Preparations of chromatin-bound proteins were prepared in triplicate and subjected to SDS-PAGE and immunoblotting with the indicated antibodies in a strain that either does or does not express Rpb3-TAP Quantification of chromatin-bound Rpb1-S2P and Rpb1-S5P is shown on the right. Mcm2 is used as a loading control.


( F , G ) A 10 - fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC + / - 200mM of HU . A high level of HU was used to be able to demonstrate robust suppression of the mec1 - S1991A phenotype . ( G ) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution .

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(F,G) A 10-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- 200mM of HU. A high level of HU was used to be able to demonstrate robust suppression of the mec1-S1991A phenotype. (G) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.


( B ) Amongst the Mec1 - S1991 - dependent phosphopeptides on HU , factors involved in transcription are highlighted .

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(B) Amongst the Mec1-S1991-dependent phosphopeptides on HU, factors involved in transcription are highlighted.


( E ) Position relative to the nuclear envelope ( Zone 1 of the lacO - tagged GAL1 - GAL10 locus in the indicated strains grown either 12h on glucose , or 90 min galactose or galactose + 0 . 2M HU . The third panel shows the relative retention at the nuclear periphery ( Zone 1 ) on galactose + HU . The number of cells is > 300 for each condition .

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(E) Position relative to the nuclear envelope (Zone 1 of the lacO-tagged GAL1-GAL10 locus in the indicated strains grown either 12h on glucose, or 90 min galactose or galactose + 0.2M HU. The third panel shows the relative retention at the nuclear periphery (Zone 1) on galactose + HU. The number of cells is >300 for each condition.


Total Rpb1 levels of exponentially growing cells that were synchronized in G1 with α - factor and released into S phase at 25 ° C . Actin was used as a loading control .

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Total Rpb1 levels of exponentially growing cells that were synchronized in G1 with α-factor and released into S phase at 25°C. Actin was used as a loading control.


A stable inducible HEK - 293T cell line expressing myc - PP2AC ( panel A were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours and myc - PP2AC affinity purified , respectively . The binding of the indicated proteins was analysed by Western blotting A ) representative of 3 independent

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A stable inducible HEK-293T cell line expressing myc-PP2AC (panel A were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2AC affinity purified, respectively. The binding of the indicated proteins was analysed by Western blotting A) representative of 3 independent experiments


A stable inducible HEK - 293T cell line expressing myc - PP2AC ( panel B ) were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours and myc - PP2AC by quantitative mass spectrometry ( B ) . B ) analysis of a biological triplicate .

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A stable inducible HEK-293T cell line expressing myc-PP2AC (panel B) were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2AC by quantitative mass spectrometry (B). B) analysis of a biological triplicate.


a stable inducible HeLa cell line expressing myc - PP2R1A ( C ) were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours and myc - PP2R1A affinity purified , respectively . The binding of the indicated proteins was analysed by Western blotting C ) representative of 2 technical

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a stable inducible HeLa cell line expressing myc- PP2R1A (C) were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2R1A affinity purified, respectively. The binding of the indicated proteins was analysed by Western blotting C) representative of 2 technical repeats


D ) HEK - 293T cells were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours and protein lysates were incubated with a microcystin affinity column to capture PPP complexes . Bound complexes were analysed by mass spectrometry to identify PP2A components . Analysis conducted on a biological triplicate .

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D) HEK-293T cells were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and protein lysates were incubated with a microcystin affinity column to capture PPP complexes. Bound complexes were analysed by mass spectrometry to identify PP2A components. Analysis conducted on a biological triplicate.


E ) Doxycyclin - inducible CRISPR - Cas9 HeLa cell lines allow for depletion of specific B56 subunits . Growth assays in the presence of the indicated concentrations of DT - 061 or iHAP1 measured after 12 days of treatment . Mean and SD of three independent experiments are shown .

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E) Doxycyclin-inducible CRISPR-Cas9 HeLa cell lines allow for depletion of specific B56 subunits. Growth assays in the presence of the indicated concentrations of DT-061 or iHAP1 measured after 12 days of treatment. Mean and SD of three independent experiments are shown.


A ) Schematic of the experimental setup and results of CRISPR screen . Genes were ranked based on their normZ score . The top hits which were further analysed are highlighted .

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A) Schematic of the experimental setup and results of CRISPR screen. Genes were ranked based on their normZ score. The top hits which were further analysed are highlighted.


B ) Validation of CRISPR screen results using RNAi depletion followed by SRB growth assay . Cells were treated with DMSO ( Ctr ) or DT - 061 5 . 5 µM or iHAP1 0 . 5 µM . C ) Incucyte growth assay after RNAi depletion of indicated proteins alone or in combination with DMSO or 5 . 5 μM DT - 061 or 0 . 5 μM iHAP1 treatment . Data information : B - C ) Mean and SD of three independent experiments is shown .

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B) Validation of CRISPR screen results using RNAi depletion followed by SRB growth assay. Cells were treated with DMSO (Ctr) or DT-061 5.5 µM or iHAP1 0.5 µM. C) Incucyte growth assay after RNAi depletion of indicated proteins alone or in combination with DMSO or 5.5 μM DT-061 or 0.5 μM iHAP1 treatment. Data information: B-C) Mean and SD of three independent experiments is shown.


A ) In vitro tubulin polymerization assay showing concentration dependent effect of iHAP1 on polymerization dynamics . Taxol and Nocodazole served as controls for the experimental setup . The assay was performed in the presence of the indicated drug concentrations . Mean and SEM are shown from three to six independent experiments .

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A) In vitro tubulin polymerization assay showing concentration dependent effect of iHAP1 on polymerization dynamics. Taxol and Nocodazole served as controls for the experimental setup. The assay was performed in the presence of the indicated drug concentrations. Mean and SEM are shown from three to six independent experiments.


B ) Representative colour - coded temporal projection of U2OS EB1 - GFP cells following indicated treatment . Scale bar 10µm .

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B) Representative colour-coded temporal projection of U2OS EB1-GFP cells following indicated treatment. Scale bar 10µm.


C ) Quantification of microtubule dynamics acquired from manual tracking of EB1 - GFP comets as indicators for MT growth after drug treatment . The mean and SD are plotted from three independent experiments ( n = 10 cells per experiment ) . Data information : P - values were calculated using Student ' s t - test or Mann - Whitney U - test ( unpaired , two - tailed ) . ns , not significant , * * * * p < 0 . 0001 .

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C) Quantification of microtubule dynamics acquired from manual tracking of EB1-GFP comets as indicators for MT growth after drug treatment. The mean and SD are plotted from three independent experiments (n = 10 cells per experiment). Data information: P-values were calculated using Student's t-test or Mann-Whitney U-test (unpaired, two-tailed). ns, not significant, ****p<0.0001.


D ) Representative temporal projection of mitotic spindles of U2OS EB1 - GFP cells following specified treatment . DNA counterstained with SiR - DNA shown in cyan in merged images . Scale bar 10µm .

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D) Representative temporal projection of mitotic spindles of U2OS EB1-GFP cells following specified treatment. DNA counterstained with SiR-DNA shown in cyan in merged images. Scale bar 10µm.


E ) Analysis of number of EB1 - GFP comets per mitotic spindle . The mean and SD are plotted from three independent experiments ( Total number of cells = 26 ( DMSO ) , 20 ( 30µM DT - 061 ) , 27 ( DMSO + APC / C in . ) and , 26 ( 2µM iHAP1 + APC / C in ) . Data information : P - values were calculated using Student ' s t - test or Mann - Whitney U - test ( unpaired , two - tailed ) . ns , not significant , * * * * p < 0 . 0001 .

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E) Analysis of number of EB1-GFP comets per mitotic spindle. The mean and SD are plotted from three independent experiments (Total number of cells = 26 (DMSO), 20 (30µM DT-061), 27 (DMSO+APC/C in.) and, 26 (2µM iHAP1+APC/C in). Data information: P-values were calculated using Student's t-test or Mann-Whitney U-test (unpaired, two-tailed). ns, not significant, ****p<0.0001.


A ) Measurement of time spent in mitosis by time - lapse imaging taking into consideration whether cells exited mitosis or underwent cell death during mitosis . n indicates the number of cells analyzed from at least 2 independent experiments . Schematic of experimental protocol on the top . Cells were treated with iHAP1 when indicated at the concentration stated in the figure . B ) Representative stills from time - lapse imaging experiments depicting the two phenotypes . Scale bar represents 20 µm .

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A) Measurement of time spent in mitosis by time-lapse imaging taking into consideration whether cells exited mitosis or underwent cell death during mitosis. n indicates the number of cells analyzed from at least 2 independent experiments. Schematic of experimental protocol on the top. Cells were treated with iHAP1 when indicated at the concentration stated in the figure. B) Representative stills from time-lapse imaging experiments depicting the two phenotypes. Scale bar represents 20 µm.


A ) Time - lapse imaging of HeLa cells expressing a marker staining the Golgi apparatus ( green ) ( n = 87 of cells from 2 experiments analysed all showing dispersal of marker ) . At time 0 , DT - 061 was added . On the right - hand side , the co - localisation of DT - 061 ( red ) and the Golgi marker is shown . B ) As in A ) but with a marker for ER and either with the addition of DT - 061 alone or DT - 061 + okadaic acid ( 50 nM ) Data information : Experiments were repeated at least twice and stills show a representative cell for each treatment condition . Cells were treated with DT - 061 at the concentration stated in the figure . A - B ) Scale bar indicates 20 µm .

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A) Time-lapse imaging of HeLa cells expressing a marker staining the Golgi apparatus (green) (n= 87 of cells from 2 experiments analysed all showing dispersal of marker). At time 0, DT-061 was added. On the right-hand side, the co-localisation of DT-061 (red) and the Golgi marker is shown. B) As in A) but with a marker for ER and either with the addition of DT-061 alone or DT-061 + okadaic acid (50 nM) Data information: Experiments were repeated at least twice and stills show a representative cell for each treatment condition. Cells were treated with DT-061 at the concentration stated in the figure. A-B) Scale bar indicates 20 µm.


C ) Time required for ER signal translocation into the nucleus after drug treatment . Number of cells analysed per condition shown on top and median indicated by red line . Data information : Experiments were repeated at least

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C) Time required for ER signal translocation into the nucleus after drug treatment. Number of cells analysed per condition shown on top and median indicated by red line. Data information: Experiments were repeated at least twice


D ) Percentage of cells with ER signal translocation into the nucleus after drug treatment . Number of cells analysed per condition shown on top . Data information : Experiments were repeated at least

D) Percentage of cells with ER signal translocation into the nucleus after drug treatment. Number of cells analysed per condition shown on top. Data information: Experiments were repeated at least twice


A - B ) Time - lapse imaging of H358 cells expressing a marker for the Golgi ( A ) or the ER ( B ) . Cells were treated with DT - 061 at time 0 . ( B , low panel ) . For A ) 177 cells were analysed from one experiment and all displayed dispersal of the Golgi marker . Arrow indicates addition of compound . Data information : Experiments were repeated at least twice except filming of the Golgi marker which was done once . Stills show a representative cell for each treatment condition . Cells were treated with DT - 061 at the concentration stated in the figure . A - B ) Scale bar indicates 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B) Time-lapse imaging of H358 cells expressing a marker for the Golgi (A) or the ER (B). Cells were treated with DT-061 at time 0. (B, low panel). For A) 177 cells were analysed from one experiment and all displayed dispersal of the Golgi marker. Arrow indicates addition of compound. Data information: Experiments were repeated at least twice except filming of the Golgi marker which was done once. Stills show a representative cell for each treatment condition. Cells were treated with DT-061 at the concentration stated in the figure. A-B) Scale bar indicates 20 µm.


C ) Time needed for ER translocation into the nucleus after drug treatment . Median ( red line ) : 160 min . Number of cells analysed per condition shown on top . Data information : Experiments were repeated at least

O O O O O O O O O O O O O O O O O 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) Time needed for ER translocation into the nucleus after drug treatment. Median (red line): 160 min. Number of cells analysed per condition shown on top. Data information: Experiments were repeated at least twice


D ) Percentage of cells with ER nuclear translocation after drug treatment . Number of cells analysed per condition shown on top . Data information : Experiments were repeated at least

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D) Percentage of cells with ER nuclear translocation after drug treatment. Number of cells analysed per condition shown on top. Data information: Experiments were repeated at least twice


B ) Increase in the molar quantity of sphingolipid classes assessed in MCF7 cells after feeding with the precursor sphingosine . MCF7 cells were co - treated with 15 μM DT - 061 , 1 . 5 μM iHAP1 , 3 µg / mL Brefeldin A , or vehicle ( DMSO ) . The determined molar quantities of sphingolipid classes were normalized to that of PC . The presented values represent averages of five independent experiments and statistical analysis was performed using an independent t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Cer : ceramide , HexCer : hexosylceramide , PC : phosphatidylcholine , and SM : sphingomyelin .

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B) Increase in the molar quantity of sphingolipid classes assessed in MCF7 cells after feeding with the precursor sphingosine. MCF7 cells were co-treated with 15 μM DT-061, 1.5 μM iHAP1, 3 µg/mL Brefeldin A, or vehicle (DMSO). The determined molar quantities of sphingolipid classes were normalized to that of PC. The presented values represent averages of five independent experiments and statistical analysis was performed using an independent t-test. *p<0.05, **p<0.01, ***p<0.001. Cer: ceramide, HexCer: hexosylceramide, PC: phosphatidylcholine, and SM: sphingomyelin.


( A ) Bone Marrow Derived Macrophages ( BMDMs ) were treated with either the AMPK activator 991 , Dex or a combination for 1 h before analysis by western blot . ( B , C ) Phospho - AMPK ( B ) and phospho - ACC ( C ) were quantified . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 6 ( B , C ) , biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical analysis using student ' s t - test ( B , C )

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(A) Bone Marrow Derived Macrophages (BMDMs) were treated with either the AMPK activator 991, Dex or a combination for 1 h before analysis by western blot. (B, C) Phospho-AMPK (B) and phospho-ACC (C) were quantified. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 6 (B,C), biological replicates * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using student's t-test (B, C)


( D ) WT and AMPKα1 - / - BMDMs were treated with Dex for 1 h before analysis by western blot and GR phosphorylation at S211 was quantified . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 3 ( D ) biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical analysis using ANOVA tests ( D ,

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(D) WT and AMPKα1-/- BMDMs were treated with Dex for 1 h before analysis by western blot and GR phosphorylation at S211 was quantified. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 3 (D) biological replicates * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests (D,


( E ) WT and AMPKα1 - / - BMDMs were treated with Dex for 24 h and analyzed by RNA - seq . Differentially expressed genes between any condition were clustered using c - means clustering .

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(E) WT and AMPKα1-/- BMDMs were treated with Dex for 24 h and analyzed by RNA-seq. Differentially expressed genes between any condition were clustered using c-means clustering.


( F ) WT and AMPKα1 - / - BMDMs were treated with Dex for 24 h and anti - / pro - inflammatory markers assessed using immunofluorescence . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 3 - 4 ( F ) biological

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(F) WT and AMPKα1-/- BMDMs were treated with Dex for 24 h and anti-/pro-inflammatory markers assessed using immunofluorescence. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 3-4 (F) biological replicates


( G WT and AMPKα1 - / - BMDMs were treated with Dex for 72 h , washed and conditioned medium was recovered after 24 h used to treat muscle stem cells . Proliferation ( Ki67 immunostaining ) ( G ) were assessed in muscle stem cells . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 4 biological replicates ( G * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical analysis using ANOVA tests Bar = 25 µm (

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(G WT and AMPKα1-/- BMDMs were treated with Dex for 72 h, washed and conditioned medium was recovered after 24 h used to treat muscle stem cells. Proliferation (Ki67 immunostaining) (G) were assessed in muscle stem cells. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 4 biological replicates (G * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests Bar=25 µm (G


H ) WT and AMPKα1 - / - BMDMs were treated with Dex for 72 h , washed and conditioned medium was recovered after 24 h used to treat muscle stem cells . differentiation ( desmin immunostaining and counting the percentage of muscle cells with 2 nuclei or more ) ( H ) were assessed in muscle stem cells . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 4 biological replicates H ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical analysis using ANOVA tests , H ) . Bar = 25 µm H ) .

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H) WT and AMPKα1-/- BMDMs were treated with Dex for 72 h, washed and conditioned medium was recovered after 24 h used to treat muscle stem cells. differentiation (desmin immunostaining and counting the percentage of muscle cells with 2 nuclei or more) (H) were assessed in muscle stem cells. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 4 biological replicates H). * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests ,H). Bar=25 µm H).


After cardiotoxin injection to damage the muscle , AMPKα1fl / fl ( WT ) and LysM - α1 - / - mice were treated with a single dose of Dexamethasone ( Dex ) intra - peritoneal ( i . p . ) ( 0 . 1 mg / kg ) at day 3 ( D3 ) and Tibialis Anterior ( TA ) muscles were harvested at day 8 ( D8 ) and 14 ( D14 ) after injury . ( A ) Representative embryonic myosin heavy chain ( EmbMHC ) immunostaining on whole TA muscle section at D8 and D14 after injury . ( B ) Quantification of ( A ) , results are expressed as percentage of positive myofibers expressing the EmbMHC on the whole muscle section . Data information : results are displayed with box and whiskers in which the central band represents the median ( B N = 7 - 12 muscles ( B * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 using ANOVA tests . Bars = 500 µm ( A ) ,

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After cardiotoxin injection to damage the muscle, AMPKα1fl/fl (WT) and LysM-α1-/- mice were treated with a single dose of Dexamethasone (Dex) intra-peritoneal (i.p.) (0.1 mg/kg) at day 3 (D3) and Tibialis Anterior (TA) muscles were harvested at day 8 (D8) and 14 (D14) after injury. (A) Representative embryonic myosin heavy chain (EmbMHC) immunostaining on whole TA muscle section at D8 and D14 after injury. (B) Quantification of (A), results are expressed as percentage of positive myofibers expressing the EmbMHC on the whole muscle section. Data information: results are displayed with box and whiskers in which the central band represents the median (B N= 7-12 muscles (B * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 using ANOVA tests. Bars = 500 µm (A),


( F ) Macrophage polarization was determined by immunofluorescence in vehicle , DAMPs and DAMPs + Dex treated WT and AMPKα1 - / - BMDMs . Data information : results are displayed with box and whiskers in which the central band represents the means ± SEM ( F ) . N = 4 - 5 biological replicates ( F ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 using ANOVA tests . Bars = 25 µm ( F ) .

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(F) Macrophage polarization was determined by immunofluorescence in vehicle, DAMPs and DAMPs+Dex treated WT and AMPKα1-/- BMDMs. Data information: results are displayed with box and whiskers in which the central band represents the means ± SEM (F). N= 4-5 biological replicates (F). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 using ANOVA tests. Bars = 25 µm (F).


( F ) Macrophage polarization was determined by immunofluorescence in vehicle , LPS and LPS + Dex treated WT and AMPKα1 - / - BMDMs , data are expressed as percentage positive cells . Data information : Results are displayed with box and whiskers in which the central band represents the means ± SEM ( F ) . N = 3 ( F ) biological replicates . Statistical analysis by ANOVA F ) * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bar = 25 µm ( F ) .

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(F) Macrophage polarization was determined by immunofluorescence in vehicle, LPS and LPS+Dex treated WT and AMPKα1-/- BMDMs, data are expressed as percentage positive cells. Data information: Results are displayed with box and whiskers in which the central band represents the means ± SEM (F). N= 3 (F) biological replicates. Statistical analysis by ANOVA F) * p<0.05, ** p<0.01, *** p<0.001. Bar = 25 µm (F).


( F ) WT and AMPKα1 - / - Bone Marrow Derived Macrophages ( BMDMs ) were treated with either Dex or the AMPK activator 991 and protein phosphorylated on serine were immunoprecipitated . Westen - blot for FOXO3 , AMPKα1 , actin and the phosphorylated form of FOXO3 were done . Quantification of the band intensity is provided normalized to those of actin and total FOXO3 , a representative example is shown . Data information : results are box and whiskers in which the central band represents the median F , N = 4 - 5 ( F biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 using ANOVA and Kruskal - Wallis

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(F) WT and AMPKα1-/- Bone Marrow Derived Macrophages (BMDMs) were treated with either Dex or the AMPK activator 991 and protein phosphorylated on serine were immunoprecipitated. Westen-blot for FOXO3, AMPKα1, actin and the phosphorylated form of FOXO3 were done. Quantification of the band intensity is provided normalized to those of actin and total FOXO3, a representative example is shown. Data information: results are box and whiskers in which the central band represents the median F, N= 4-5 (F biological replicates. * p<0.05, ** p<0.01, *** p<0.001 **** p<0.0001 using ANOVA and Kruskal-Wallis tests