( L ) Histogram confirming the decreased number of Tuj1 + neurons in the ventral , but not dorsal , retina of Nr2f1 mutants . data are represented as mean ± SEM ; N = 3 - 4 Student t - test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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

(L) Histogram confirming the decreased number of Tuj1+ neurons in the ventral, but not dorsal, retina of Nr2f1 mutants. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


( M - N ' ) Double Tuj1 / Brn3a IF on E13 . 5 WT and KO optic cup sections revealing decreased numbers of differentiating RGCs in bending ventral retinae of KO embryos ( N ' ) .

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

(M-N') Double Tuj1/Brn3a IF on E13.5 WT and KO optic cup sections revealing decreased numbers of differentiating RGCs in bending ventral retinae of KO embryos (N').


( O , O ' ) Details of P28 WT and HET retinae indicating decreased number of Brn3a + neurons ( arrowheads ) in the GCL of HET animals .

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

(O,O') Details of P28 WT and HET retinae indicating decreased number of Brn3a+ neurons (arrowheads) in the GCL of HET animals.


( P ) Graph illustrating the dynamics of RGC differentiation from E13 . 5 to P28 , normalized to WT animals . data are represented as mean ± SEM ; N = 3 - 4 Student t - test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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

(P) Graph illustrating the dynamics of RGC differentiation from E13.5 to P28, normalized to WT animals. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


Q - Q " ) Cross - sections of DiI - labelled P0 ONs in WT and KO

Q-Q") Cross-sections of DiI-labelled P0 ONs in WT and KO pups


( R , R ' ) Tuj1 + P8 ON fibers in WT and KO pups showing a progressive volume

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

(R,R') Tuj1+ P8 ON fibers in WT and KO pups showing a progressive volume loss


( S ) Quantification of the surface occupied by Tuj1 + or DiI + fibers in different genotypes and ages , as indicated . data are represented as mean ± SEM ; N = 4 - 5 Student t - test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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

(S) Quantification of the surface occupied by Tuj1+ or DiI+ fibers in different genotypes and ages, as indicated. data are represented as mean ± SEM; N=4-5 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


( T - U ' ) Cleaved - Caspase - 3 ( green , apoptotic cells ) and Brn3a ( red , RGCs ) IF on transverse sections of E18 . 5 retinae in WT and KO fetuses . High magnifications ( T ' - T ' " ) highlight an example of a double - labeled RGC undergoing apoptosis ( arrowheads ) in the ganglion cell layer ( GCL ) . Apoptotic RGCs are increased in KO animals ( arrowheads in U ' ) , compared to WT ( U ) .

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

(T-U') Cleaved-Caspase-3 (green, apoptotic cells) and Brn3a (red, RGCs) IF on transverse sections of E18.5 retinae in WT and KO fetuses. High magnifications (T'-T'") highlight an example of a double-labeled RGC undergoing apoptosis (arrowheads) in the ganglion cell layer (GCL). Apoptotic RGCs are increased in KO animals (arrowheads in U'), compared to WT (U).


( V ) Histogram quantifying the number of RGC apoptotic cells in WT , HET and KO from E18 . 5 to P5 . Note the significant increase of dying cells at E18 . 5 . Nuclei ( blue ) were stained with DAPI . data are represented as mean ± SEM ; N = 3 - 4 Student t - test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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

(V) Histogram quantifying the number of RGC apoptotic cells in WT, HET and KO from E18.5 to P5. Note the significant increase of dying cells at E18.5. Nuclei (blue) were stained with DAPI. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


( A - F ) Pax2 ( magenta , astrocytic progenitors ) and Tuj1 ( green , RGC axons ) IF on E13 . 5 , E15 . 5 and E18 . 5 OS / ONs cross - sections showing an increase of Pax2 - expressing cells within the optic nerve ( ON ) of KO embryos at all stages .

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

(A-F) Pax2 (magenta, astrocytic progenitors) and Tuj1 (green, RGC axons) IF on E13.5, E15.5 and E18.5 OS/ONs cross-sections showing an increase of Pax2-expressing cells within the optic nerve (ON) of KO embryos at all stages.


Histograms quantifying the average number of Pax2 + ( G , H ) cells in WT , HET and KO at different stages and axial length of the ON , as indicated . Astrocytic progenitors are significantly increased along the nerve of mutants . data are represented as means ± SEM ; N = 3 - 5 . Statistical significance was obtained by Student t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars :

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

Histograms quantifying the average number of Pax2+ (G,H) cells in WT, HET and KO at different stages and axial length of the ON, as indicated. Astrocytic progenitors are significantly increased along the nerve of mutants. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm


Histograms quantifying the average number of NF1A + ( I ) cells in WT , HET and KO at different stages and axial length of the ON , as indicated . Astrocytic progenitors are significantly increased along the nerve of mutants . data are represented as means ± SEM ; N = 3 - 5 . Statistical significance was obtained by Student t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars :

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

Histograms quantifying the average number of NF1A+ (I) cells in WT, HET and KO at different stages and axial length of the ON, as indicated. Astrocytic progenitors are significantly increased along the nerve of mutants. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm


( J , K ) NF1A ( red , astrocyte precursors ) IF on P7 WT and KO ON cross - sections illustrating a high astrocytic density in KO ON ( delineated by thin dashed lines ) . ( L ) Histogram confirming the increased percentage of NF1A + astrocytes in HET and KO ONs compared to WT . data are represented as means ± SEM ; N = 3 - 5 . Statistical significance was obtained by Student t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars :

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

(J,K) NF1A (red, astrocyte precursors) IF on P7 WT and KO ON cross-sections illustrating a high astrocytic density in KO ON (delineated by thin dashed lines). (L) Histogram confirming the increased percentage of NF1A+ astrocytes in HET and KO ONs compared to WT. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm


( M - O " ) Tuj1 ( green , RGC axons ) and S100β ( red , astrocytes ) IF on P7 WT , HET and KO ONs . Arrowheads point to S100β + astrocyte groups ( N ' , O ' ) and absence of Tuj1 + fibers ( N ' ' , O ' ' ) in HET and KO ONs .

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

(M-O") Tuj1 (green, RGC axons) and S100β (red, astrocytes) IF on P7 WT, HET and KO ONs. Arrowheads point to S100β+ astrocyte groups (N',O') and absence of Tuj1+ fibers (N'',O'') in HET and KO ONs.


( P . Q ) TEM images of WT and KO P8 ONs showing thick astrocytic processes filled with intermediate filaments ( red dotted lines and arrowheads ) in KO nerves . Scale bars ( 500nm )

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

(P.Q) TEM images of WT and KO P8 ONs showing thick astrocytic processes filled with intermediate filaments (red dotted lines and arrowheads) in KO nerves. Scale bars (500nm)


( R - S ' ) S100β ( green , astrocytes ) and Sox2 ( red , reactive / inflamed astrocytes ) IF on P28 WT and HET ONs illustrating strong Sox2 expression in reactive astrocytes .

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(R-S') S100β (green, astrocytes) and Sox2 (red, reactive/inflamed astrocytes) IF on P28 WT and HET ONs illustrating strong Sox2 expression in reactive astrocytes.


( T ) Graph quantifying the average number of Sox2 + astrocytes per P28 ON section in KO and WT animals . data are represented as means ± SEM ; N = 3 - 5 . Statistical significance was obtained by Student t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars :

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

(T) Graph quantifying the average number of Sox2+ astrocytes per P28 ON section in KO and WT animals. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm


( U , V ) EM sections of P28 WT and HET ONs illustrating the expanded astrocytic processes ( surrounded by red dotted lines ) among myelinated fibers of P28 ONs . Scale bars : ( U , V ) ( 4µm ) .

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

(U,V) EM sections of P28 WT and HET ONs illustrating the expanded astrocytic processes (surrounded by red dotted lines) among myelinated fibers of P28 ONs. Scale bars: (U,V) (4µm).


( A - C ) NF1A ( red , astrocytes ) and Sox10 ( green , oligodendrocytes ) IF on cross - sections of WT and KO P8 ONs showing altered ratio of astrocytes and oligodendrocytes in mutants , particularly in KOs , as quantified in ( C ) . data are represented as means ± SEM . N = 4 - 5 Statistical significance was obtained by Student t - test or by two - way ANOVA when comparing 2 or multiple conditions , respectively ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Scale bars :

O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) NF1A (red, astrocytes) and Sox10 (green, oligodendrocytes) IF on cross-sections of WT and KO P8 ONs showing altered ratio of astrocytes and oligodendrocytes in mutants, particularly in KOs, as quantified in (C). data are represented as means ± SEM. N=4-5 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001 Scale bars: 50µm


( D - J ) MBP ( yellow , fully differentiated oligodendrocytes ) IF on cross - sections of WT , HET and KO P7 optic nerves ( ONs ) at the chiasm ( D - F ) and between the chiasm and the eye ( G - I ) . Note the dramatic decrease of MBP + oligodendrocytes at the chiasm in HET and KO , quantified in ( J ) . The ratio of MBP + cells is calculated over the total DAPI + cell number per chiasmal / ON section . data are represented as means ± SEM . N = 4 - 5 Statistical significance was obtained by Student t - test or by two - way ANOVA when comparing 2 or multiple conditions , respectively ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars :

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

(D-J) MBP (yellow, fully differentiated oligodendrocytes) IF on cross-sections of WT, HET and KO P7 optic nerves (ONs) at the chiasm (D-F) and between the chiasm and the eye (G-I). Note the dramatic decrease of MBP+ oligodendrocytes at the chiasm in HET and KO, quantified in (J). The ratio of MBP+ cells is calculated over the total DAPI+ cell number per chiasmal/ON section. data are represented as means ± SEM. N=4-5 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm


( K - N ) EM thin sections of P8 WT , HET and KO ONs depicting myelin as a dense , dark staining around axonal fibers illustrate a significantly lower number of myelinated fibers in HET and KO , as quantified in ( N ) . Higher magnification images ( insets ) showing different degrees of myelination in HET and KO ONs . data are represented as means ± SEM . N = 3 Statistical significance was obtained by Student t - test or by two - way ANOVA when comparing 2 or multiple conditions , respectively ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm ( 4µm )

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

(K-N) EM thin sections of P8 WT, HET and KO ONs depicting myelin as a dense, dark staining around axonal fibers illustrate a significantly lower number of myelinated fibers in HET and KO, as quantified in (N). Higher magnification images (insets) showing different degrees of myelination in HET and KO ONs. data are represented as means ± SEM. N=3 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm (4µm)


TEM images of P28 WT and HET confirming persistent hypomyelination in mutants at P28 Scale bars : ( 4µm )

TEM images of P28 WT and HET confirming persistent hypomyelination in mutants at P28 Scale bars: (4µm)


P28 WT and HET confirming persistent hypomyelination in mutants at P28 , as quantified in ( Q ) . data are represented as means ± SEM . N = 3 Statistical significance was obtained by Student t - test or by two - way ANOVA when comparing 2 or multiple conditions , respectively ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars :

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

P28 WT and HET confirming persistent hypomyelination in mutants at P28, as quantified in (Q). data are represented as means ± SEM. N=3 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm


( R , S ) High magnification EM images of P28 WT and HET ONs showing the difference in myelin compaction between WT ( arrowheads in R ) and HET ( arrowheads in S ) . Scale bars : ( 1µm ) .

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

(R,S) High magnification EM images of P28 WT and HET ONs showing the difference in myelin compaction between WT (arrowheads in R) and HET (arrowheads in S). Scale bars: (1µm).


( A ) Experimental design to treat HET pups from P2 to P8 with Miconazole . DMSO ( controls ) and Miconazole - treated pups were sacrificed at P8 or at P28 to evaluate the ON myelination . ( B - E ) Sox10 ( red , oligodendrocyte precursors ) and MBP ( green , fully differentiated oligodendrocytes ) IF on cross - sections of P8 HET ONs after 6 days of treatment with DMSO ( D ) or Miconazole ( E ) . Miconazole increased the number of Sox10 + cells compared to HETs treated with DMSO ( quantified in B ) and rescued MBP staining pixel intensity of HET pups to level comparable to those of WT pups ( quantified in C ) . Nuclei ( blue ) were stained with DAPI . , the error bars represent the SEM of the means ; N = 3 Statistical significance was obtained by ANOVA ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Scale bars :

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

(A) Experimental design to treat HET pups from P2 to P8 with Miconazole. DMSO (controls) and Miconazole-treated pups were sacrificed at P8 or at P28 to evaluate the ON myelination. (B-E) Sox10 (red, oligodendrocyte precursors) and MBP (green, fully differentiated oligodendrocytes) IF on cross-sections of P8 HET ONs after 6 days of treatment with DMSO (D) or Miconazole (E). Miconazole increased the number of Sox10+ cells compared to HETs treated with DMSO (quantified in B) and rescued MBP staining pixel intensity of HET pups to level comparable to those of WT pups (quantified in C). Nuclei (blue) were stained with DAPI. , the error bars represent the SEM of the means; N=3 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm


( F - I ) Sox10 ( oligodendrocyte marker , red ) and Phospho - ERK ( dually phosphorylated forms of active ERK1 and ERK2 , green ) IF in WT and HET ONs , treated with DMSO ( F ) or Miconazole ( G ) between P2 and P8 . As previously reported , Miconazole strongly activate ERK signaling pathway ( G ) . Signal intensity quantification in whole ON area is shown in ( H ) , while specific evaluation of pixel intensity around Sox10 + nuclei is shown in ( I ) . Nuclei ( blue ) were stained with DAPI . the error bars represent the SEM of the means N = 2 Statistical significance was obtained by ANOVA ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Scale bars :

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

(F-I) Sox10 (oligodendrocyte marker, red) and Phospho-ERK (dually phosphorylated forms of active ERK1 and ERK2, green) IF in WT and HET ONs, treated with DMSO (F) or Miconazole (G) between P2 and P8. As previously reported, Miconazole strongly activate ERK signaling pathway (G). Signal intensity quantification in whole ON area is shown in (H), while specific evaluation of pixel intensity around Sox10+ nuclei is shown in (I). Nuclei (blue) were stained with DAPI. the error bars represent the SEM of the means N=2 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm


( J - M ) EM thin sections of P28 WT and HET ONs , treated with DMSO ( J , K ) or Miconazole ( L ) between P2 and P8 . Different degrees of myelin compaction can be appreciated at high magnification , with HET ONs showing non compacted sheaths ( white arrows in K ) and vacuoles ( yellow arrows ) . Miconazole treatment rescues almost normal myelin g - ratio , as quantified in M . Nuclei ( blue ) were stained with DAPI . the error bars represent the SEM of the means N = 2 Statistical significance was obtained by ANOVA ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Scale bars : 50µm , except ( J - L ) ( 500nm ) .

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

(J-M) EM thin sections of P28 WT and HET ONs, treated with DMSO (J,K) or Miconazole (L) between P2 and P8. Different degrees of myelin compaction can be appreciated at high magnification, with HET ONs showing non compacted sheaths (white arrows in K) and vacuoles (yellow arrows). Miconazole treatment rescues almost normal myelin g-ratio, as quantified in M. Nuclei (blue) were stained with DAPI. the error bars represent the SEM of the means N=2 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm, except (J-L) (500nm).


( N - P ' ) S100β ( green , astrocytes ) and Sox2 ( red , reactive / proliferative astrocytes ) IF in the ONs of WT ( N , N ' ) and HET ( O - P ' ) animals after 6 days of treatment with DMSO ( N - O ' ) or Miconazole ( P , P ' ) , then sacrificed at P28 . Astrocytes undergo remodeling and express Sox2 in Nr2f1 HET animals ( O ' , P ' ) , even after Miconazole treatment .

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

(N-P') S100β (green, astrocytes) and Sox2 (red, reactive/proliferative astrocytes) IF in the ONs of WT (N,N') and HET (O-P') animals after 6 days of treatment with DMSO (N-O') or Miconazole (P,P'), then sacrificed at P28. Astrocytes undergo remodeling and express Sox2 in Nr2f1 HET animals (O',P'), even after Miconazole treatment.


Histogram showing the average number of S100β + / Sox2 + astrocytes per P28 ON section in WT or HET animals treated with DMSO or Miconazole as indicated , with Sox2 high ( magenta columns ) and low levels ( pink columns ) . Nuclei ( blue ) were stained with DAPI . the error bars represent the SEM of the means ; ; N = 2 Statistical significance was obtained by ANOVA ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Scale bars :

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Histogram showing the average number of S100β+/Sox2+ astrocytes per P28 ON section in WT or HET animals treated with DMSO or Miconazole as indicated, with Sox2 high (magenta columns) and low levels (pink columns). Nuclei (blue) were stained with DAPI. the error bars represent the SEM of the means; ; N=2 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm


( B ) Nissl - stained sections illustrating the location of the recording electrodes ( arrows ) . dhc , dorsal hippocampal commissure ; PAC , periaqueductal grey ; Th , thalamus .

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(B) Nissl-stained sections illustrating the location of the recording electrodes (arrows). dhc, dorsal hippocampal commissure; PAC, periaqueductal grey; Th, thalamus.


( C ) Examples of field potentials ( averaged ≥ 20 times ) evoked by flash stimulation in the recording sites for WT and HET mice . Dotted lines indicate the beginning of evoked field potentials .

(C) Examples of field potentials (averaged ≥ 20 times) evoked by flash stimulation in the recording sites for WT and HET mice. Dotted lines indicate the beginning of evoked field potentials.


( D ) Latency of evoked field potentials for WT ( N = 13 ) and HET ( N = 18 ) mice .

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(D) Latency of evoked field potentials for WT (N=13) and HET (N=18) mice.


( E ) In a first series of experiments , animals [ WT ( N = 13 ) and HET ( N = 18 ) ] were trained to press a lever to obtain a pellet of food in an illuminated Skinner box with a fixed - ratio ( 1 : 1 ) schedule . After ten days of training , they were transferred to a light on / light off protocol where lever presses were only rewarded when a small light bulb located over the lever was switched on . Lever presses while the bulb was off were punished with a time penalty of ≤ 10 s during which the bulb would not turn on . ( F , G ) Mean days to criterion ( F ) and mean number of lever presses during the last two training sessions ( G ) for the fixed - ratio ( 1 : 1 ) paradigm . No significant differences were observed .

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(E) In a first series of experiments, animals [WT (N=13) and HET (N=18)] were trained to press a lever to obtain a pellet of food in an illuminated Skinner box with a fixed-ratio (1:1) schedule. After ten days of training, they were transferred to a light on/light off protocol where lever presses were only rewarded when a small light bulb located over the lever was switched on. Lever presses while the bulb was off were punished with a time penalty of ≤ 10 s during which the bulb would not turn on. (F,G) Mean days to criterion (F) and mean number of lever presses during the last two training sessions (G) for the fixed-ratio (1:1) paradigm. No significant differences were observed.


( H ) Performance of WT ( N = 13 ) and HET ( N = 18 ) mice in the light on / light off test . Criterion ( dotted line ) was that the animal had to press the lever more times during the small bulb light on period than during the light off one . WT mice performed better than HET animals and reached the criterion by the 6th session . The HET did not reached criterion for the duration of the test . While a linear fit to WT data increased significantly across training , the same linear fit to HET performance did not present any significant change .

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(H) Performance of WT (N=13) and HET (N=18) mice in the light on/light off test. Criterion (dotted line) was that the animal had to press the lever more times during the small bulb light on period than during the light off one. WT mice performed better than HET animals and reached the criterion by the 6th session. The HET did not reached criterion for the duration of the test. While a linear fit to WT data increased significantly across training, the same linear fit to HET performance did not present any significant change.


( I ) The percentage of lever presses during the light on and light off periods for WT and HET mice was also different , because the WT group pressed the lever preferentially during the light period . Data were averaged from the 9th and 10th sessions . For statistical significance ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(I) The percentage of lever presses during the light on and light off periods for WT and HET mice was also different, because the WT group pressed the lever preferentially during the light period. Data were averaged from the 9th and 10th sessions. For statistical significance (*P<0.05; **P<0.01; ***P<0.001).


Human first - trimester trophoblast cell line Sw . 71 ( left panel ) and human primary culture ( right panel ) trophoblast cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media over time . RNA was collected for measuring viral titers by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates . NT , no treatment group ; HPC , human primary culture . ( A ) ZIKV titers in Sw . 71 and HPC trophoblasts . * p < 0 . 05 by One - way ANOVA .

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Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (A) ZIKV titers in Sw.71 and HPC trophoblasts. *p < 0.05 by One-way ANOVA.


Human first - trimester trophoblast cell line Sw . 71 ( left panel ) and human primary culture ( right panel ) trophoblast cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media over time . RNA was collected for measuring gene expression by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates . NT , no treatment group ; HPC , human primary culture . ( B ) IFNα is not highly induced in response to ZIKV infection in trophoblast cells . IFNα mRNA expression was inhibited in the first 24 h . p . i . and maintained at low level during ZIKV infection . Difference not significant ( n . s . ) by One - way ANOVA .

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Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (B) IFNα is not highly induced in response to ZIKV infection in trophoblast cells. IFNα mRNA expression was inhibited in the first 24 h.p.i. and maintained at low level during ZIKV infection. Difference not significant (n.s.) by One-way ANOVA.


Human first - trimester trophoblast cell line Sw . 71 ( left panel ) and human primary culture ( right panel ) trophoblast cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media over time . RNA was collected for measuring gene expression by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates . NT , no treatment group ; HPC , human primary culture . ( C ) ZIKV infection induced a time - dependent increase in IFNβ mRNA expression in Sw . 71 and HPC . * p < 0 . 05 , * * p < 0 . 01 by One - way ANOVA .

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Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (C) ZIKV infection induced a time-dependent increase in IFNβ mRNA expression in Sw.71 and HPC. *p < 0.05, **p < 0.01 by One-way ANOVA.


Human first - trimester trophoblast cell line Sw . 71 ( left panel ) and human primary culture ( right panel ) trophoblast cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media over time . ( D ) Expression levels of secreted IFNβ detected in trophoblast supernatants by ELLA assay . Supernatants were collected from ZIKV - infected and control trophoblast cultures at different times , and IFNβ protein secretion was quantified by ELLA assay . Note the increase of secreted IFNβ in the ZIKV - infected supernatant in a time - dependent manner . * * * * p < 0 . 0001 by Student ' s t - test against individual time point NT control .

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

Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. (D) Expression levels of secreted IFNβ detected in trophoblast supernatants by ELLA assay. Supernatants were collected from ZIKV-infected and control trophoblast cultures at different times, and IFNβ protein secretion was quantified by ELLA assay. Note the increase of secreted IFNβ in the ZIKV-infected supernatant in a time-dependent manner. ****p < 0.0001 by Student's t-test against individual time point NT control.


Sw . 71 ( left panel ) and HPC cells ( right panel ) were treated with IFNβ to detect ISG20 mRNA expression . Data represent as mean ± SEM ; n = 3 biological replicates . NT , no treatment group ; HPC , human primary culture . ( A ) Sw . 71 and HPC cells were treated with different doses of IFNβ ( 3 , 30 , 300 ng / ml ) for 8 h and RNA were collected for determining ISG20 mRNA expressions by qRT - PCR . Note the increase of ISG20 mRNA expression in a dose - dependent manner . * p < 0 . 05 , * * p < 0 . 01 by One - way ANOVA . ( B ) Sw . 71 and HPC cells were treated with 300ng / ml IFNβ over time and RNA was collected for determining ISG20 mRNA expressions by qRT - PCR . Note the increase of ISG20 mRNA expression in a time - dependent manner . * p < 0 . 05 by One - way ANOVA . ( C )

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Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 mRNA expression. Data represent as mean ± SEM; n=3 biological replicates. NT, no treatment group; HPC, human primary culture. (A) Sw.71 and HPC cells were treated with different doses of IFNβ (3, 30, 300 ng/ml) for 8 h and RNA were collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a dose-dependent manner. *p < 0.05, **p < 0.01 by One-way ANOVA. (B) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and RNA was collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a time-dependent manner. *p < 0.05 by One-way ANOVA. (C)


Sw . 71 ( left panel ) and HPC cells ( right panel ) were treated with IFNβ to detect ISG20 protein expression . ( D ) Sw . 71 and HPC cells were treated with 300ng / ml IFNβ over time and protein was collected for determining ISG20 protein expressions by western blot . Note that there was no ISG20 protein expression in the no treatment group in both Sw . 71 and HPC , only after IFNβ treatment , ISG20 protein expression exhibited a time - dependent manner . β - actin served as a loading control .

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Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 protein expression. (D) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and protein was collected for determining ISG20 protein expressions by western blot. Note that there was no ISG20 protein expression in the no treatment group in both Sw.71 and HPC, only after IFNβ treatment, ISG20 protein expression exhibited a time-dependent manner. β-actin served as a loading control.


( B ) No ISG20 protein expression in ISG20 - / - Sw . 71 in ZIKV infection . Sw . 71 and ISG20 - / - Sw . 71 cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media for 48 h , and proteins were collected for western blot analysis . β - actin served as a loading control .

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(B) No ISG20 protein expression in ISG20-/- Sw.71 in ZIKV infection. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and proteins were collected for western blot analysis. β-actin served as a loading control.


( C ) Higher ZIKA titer was shown in ISG20 - / - Sw . 71 cells . Sw . 71 and ISG20 - / - Sw . 71 cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media for 48 h , and RNA was collected for determining the viral titer and gene expression by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates ; * p < 0 . 05 by Student ' s t - test .

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(C) Higher ZIKA titer was shown in ISG20-/- Sw.71 cells. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and RNA was collected for determining the viral titer and gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.


( D ) More viral shedding in ZIKV - infected ISG20 - / - Sw . 71 cell culture supernatant . Supernatant from ZIKV - infected Sw . 71 and ISG20 - / - Sw . 71 cells were collected and plaque assay was performed using Vero cells . A representative plaque assay picture is presented . Note that more plaques were formed in Vero cells by incubating with ZIKV - infected ISG20 - / - Sw . 71 cell culture supernatant . Scale bar = 130μm . ( E ) Plaques were counted and plaque forming - unit ( pfu ) ratio was calculated for the comparison of the viral titers . Data represent as mean ± SEM ; n = 3 biological replicates . * p < 0 . 05 by Student ' s t - test .

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(D) More viral shedding in ZIKV-infected ISG20-/- Sw.71 cell culture supernatant. Supernatant from ZIKV-infected Sw.71 and ISG20-/- Sw.71 cells were collected and plaque assay was performed using Vero cells. A representative plaque assay picture is presented. Note that more plaques were formed in Vero cells by incubating with ZIKV-infected ISG20-/- Sw.71 cell culture supernatant. Scale bar=130μm. (E) Plaques were counted and plaque forming-unit (pfu) ratio was calculated for the comparison of the viral titers. Data represent as mean ± SEM; n=3 biological replicates. *p < 0.05 by Student's t-test.


( B ) ISGs mRNA expression stimulated by IFNβ in Sw . 71 and ISG20 - / - Sw . 71 trophoblast cells . Sw . 71 and ISG20 - / - Sw . 71 cells were treated with 30 ng / ml IFNβ for 24 h , and RNA was collected for determining the ISGs gene expression by qRT - PCR . Data represent as mean ± SEM ; n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 by Student ' s t - test against control .

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(B) ISGs mRNA expression stimulated by IFNβ in Sw.71 and ISG20-/- Sw.71 trophoblast cells. Sw.71 and ISG20-/- Sw.71 cells were treated with 30 ng/ml IFNβ for 24 h, and RNA was collected for determining the ISGs gene expression by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; *p < 0.05, **p < 0.01 by Student's t-test against control.


( D ) IFNβ pre - treatment significantly prevented trophoblast cells from ZIKV infection , however , this protection was evidently attenuated due to lack of ISG20 . Sw . 71 and ISG20 - / - Sw . 71 cells were pre - treated with or without 30 ng / ml IFNβ for 24 h , followed by ZIKV infection ( MOI = 2 ) for 1 h and refreshed with regular media for 24 h , and RNA was collected to determine the viral titers by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates ; * p < 0 . 05 by Student ' s t - test .

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(D) IFNβ pre-treatment significantly prevented trophoblast cells from ZIKV infection, however, this protection was evidently attenuated due to lack of ISG20. Sw.71 and ISG20-/- Sw.71 cells were pre-treated with or without 30 ng/ml IFNβ for 24 h, followed by ZIKV infection (MOI=2) for 1 h and refreshed with regular media for 24 h, and RNA was collected to determine the viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.


( D ) ISG20 secretion in the positive clone supernatant significantly decreased ZIKV infection in trophoblast cells . Supernatants from negative and positive clones were collected and added to ISG20 - / - Sw . 71 trophoblast cells together with ZIKA virus ( MOI = 2 ) for 1 h , followed by refreshing with new growth media for 48 h . RNA was then collected to determine viral titers by qRT - PCR . Data represent as mean ± SEM ; n = 3 biological replicates ; * * p < 0 . 01 by Student ' s t - test .

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(D) ISG20 secretion in the positive clone supernatant significantly decreased ZIKV infection in trophoblast cells. Supernatants from negative and positive clones were collected and added to ISG20-/- Sw.71 trophoblast cells together with ZIKA virus (MOI=2) for 1 h, followed by refreshing with new growth media for 48 h. RNA was then collected to determine viral titers by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01 by Student's t-test.


( E ) Recombinant ISG20 - Fc degrades ZIKA viral RNA and HSV2 viral DNA . 50ng purified viral RNA ( ZIKV ) or DNA ( HSV - 2 ) were incubated with increasing concentrations of rISG20 - Fc ( 5 , 50 , 500 ng ) in the presence of RNase inhibitor for 90min at 37 ° C followed by quantification of viral titers by qRT - PCR , and agarose gel was used to evaluate the RNA degradation by electrophoresis . The representative picture of agarose gel is presented . rISG20 - Fc was able to degrade both ZIKV RNA and HSV - 2 DNA in a dose dependent manner ; however , rISG20 - Fc was more efficient in degrading viral RNA than DNA . Data represent as mean ± SEM ; n = 3 biological replicates ; * * p < 0 . 01 . * * * p < 0 . 001 and * * * * p < 0 . 0001 by One - way ANOVA .

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(E) Recombinant ISG20-Fc degrades ZIKA viral RNA and HSV2 viral DNA. 50ng purified viral RNA (ZIKV) or DNA (HSV-2) were incubated with increasing concentrations of rISG20-Fc (5, 50, 500 ng) in the presence of RNase inhibitor for 90min at 37°C followed by quantification of viral titers by qRT-PCR, and agarose gel was used to evaluate the RNA degradation by electrophoresis. The representative picture of agarose gel is presented. rISG20-Fc was able to degrade both ZIKV RNA and HSV-2 DNA in a dose dependent manner; however, rISG20-Fc was more efficient in degrading viral RNA than DNA. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01. ***p < 0.001 and ****p < 0.0001 by One-way ANOVA.


( B ) Macroscopic evaluation of the pregnant uterus and fetus . Compare to the fetal death and reabsorptions in ZIKV - infected mice , rISG20 - Fc treatment rescued this phenotype by decreasing pregnancy loss ( resorption ) .

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(B) Macroscopic evaluation of the pregnant uterus and fetus. Compare to the fetal death and reabsorptions in ZIKV-infected mice, rISG20-Fc treatment rescued this phenotype by decreasing pregnancy loss (resorption).


( A ) rISG20 - Fc treatment alleviates maternal viral burden . Note that rISG20 - Fc treatment significantly decreased the maternal serum viral titers . Data represent as mean ± SEM ; n = 3 mice for control PBS group , and n = 4 mice for treatment groups ; * p < 0 . 05 by Student ' s t - test . ( B ) rISG20 - Fc treatment reduces ZIKV titers in the fetal brain . Although there was no difference of ZIKV titer in the placenta , at the fetal side , there was a significant decrease of viral titer in the fetal brain , suggesting rISG20 - Fc can block viral transmission from placenta to fetus . Data represent as mean ± SEM ; n = 4 for each group , and 3 - 4 placentas / fetal brains from every mouse were analyzed ; * p < 0 . 05 by Student ' s t - test . (

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(A) rISG20-Fc treatment alleviates maternal viral burden. Note that rISG20-Fc treatment significantly decreased the maternal serum viral titers. Data represent as mean ± SEM; n=3 mice for control PBS group, and n=4 mice for treatment groups; *p < 0.05 by Student's t-test. (B) rISG20-Fc treatment reduces ZIKV titers in the fetal brain. Although there was no difference of ZIKV titer in the placenta, at the fetal side, there was a significant decrease of viral titer in the fetal brain, suggesting rISG20-Fc can block viral transmission from placenta to fetus. Data represent as mean ± SEM; n=4 for each group, and 3-4 placentas/fetal brains from every mouse were analyzed; *p < 0.05 by Student's t-test. (


( C ) rISG20 - Fc treatment decreases Eotaxin significantly in maternal circulation . Data represent as mean ± SEM ; n = 2 for PBS control group , and n = 3 for every treatment group ; * p < 0 . 05 by One - way ANOVA ; difference not significant for IL - 9 and IFNγ by One - way ANOVA .

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(C) rISG20-Fc treatment decreases Eotaxin significantly in maternal circulation. Data represent as mean ± SEM; n=2 for PBS control group, and n=3 for every treatment group; *p < 0.05 by One-way ANOVA; difference not significant for IL-9 and IFNγ by One-way ANOVA.


( A ) RT - qPCR of early passage RMS tumoroid models shows positivity for at least one gene used in standard - of - care pathology analysis ( DES , MYOG , or MYOD1 ) . Conventional RMS cell lines ( RD and RH30 ) were used as positive controls , while two Synovial Sarcoma ( SS000DAZ and SS077 ) tumoroid models were used as negative controls . Gene expression was normalized to the expression of a house - keeping gene and human reference RNA ( HREF ) via the ΔΔCq method . Each tumoroid line was measured once with four technical replicates with the error bars representing the standard deviation of said technical replicates .

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(A) RT-qPCR of early passage RMS tumoroid models shows positivity for at least one gene used in standard-of-care pathology analysis (DES, MYOG, or MYOD1). Conventional RMS cell lines (RD and RH30) were used as positive controls, while two Synovial Sarcoma (SS000DAZ and SS077) tumoroid models were used as negative controls. Gene expression was normalized to the expression of a house-keeping gene and human reference RNA (HREF) via the ΔΔCq method. Each tumoroid line was measured once with four technical replicates with the error bars representing the standard deviation of said technical replicates.


( C ) Morphological ( via H & E ) and immunohistochemical ( IHC ) comparison of RMS tumors and derived RMS tumoroid models shows retained marker protein ( Desmin , Myogenin and MYOD1 ) expression and cellular morphology . Scale bars equal 200 µm ( RMS012 , RMS102 ) or 100 µm ( RMS000HQC ) .

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(C) Morphological (via H&E) and immunohistochemical (IHC) comparison of RMS tumors and derived RMS tumoroid models shows retained marker protein (Desmin, Myogenin and MYOD1) expression and cellular morphology. Scale bars equal 200 µm (RMS012, RMS102) or 100 µm (RMS000HQC).


( A ) Copy number frequency plots of RMS tumors ( upper row ) and derived RMS tumoroid models ( lower row ) divided by fusion - status ( columns ) . Chromosomes are annotated on the x - axis from left to right while the y - axis shows the percentage of samples in this group carrying a gain ( red ) or loss ( blue ) in this genomic region .

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(A) Copy number frequency plots of RMS tumors (upper row) and derived RMS tumoroid models (lower row) divided by fusion-status (columns). Chromosomes are annotated on the x-axis from left to right while the y-axis shows the percentage of samples in this group carrying a gain (red) or loss (blue) in this genomic region.


( B ) Contribution of somatic mutational signatures per tumor and tumoroid model . SBS = single base substitution , TMZ = temozolomide , T = tumor , O = tumoroid .

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(B) Contribution of somatic mutational signatures per tumor and tumoroid model. SBS = single base substitution, TMZ = temozolomide, T = tumor, O = tumoroid.


( C ) Table depicting pathogenic single - nucleotide variants ( SNVs ) in RMS tumors ( T ) and tumoroid models ( O ) . Circle color indicates SNV type while circle size indicates variant allele fraction ( VAF ) . Vertical dotted lines separate samples derived from individual patients . Highlighted are genes previously reported for this RMS subtype .

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(C) Table depicting pathogenic single-nucleotide variants (SNVs) in RMS tumors (T) and tumoroid models (O). Circle color indicates SNV type while circle size indicates variant allele fraction (VAF). Vertical dotted lines separate samples derived from individual patients. Highlighted are genes previously reported for this RMS subtype.


( D ) Correlogram of bulk mRNA sequencing expression profiles of pediatric kidney tumors ( controls ) as well as RMS tumoroid models and RMS tumors . CCRCC = Clear Cell Renal Cell Carcinoma , CMN = Congenital Mesoblastic Nephroma , WT = Wilms Tumor , Cor = correlation .

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(D) Correlogram of bulk mRNA sequencing expression profiles of pediatric kidney tumors (controls) as well as RMS tumoroid models and RMS tumors. CCRCC = Clear Cell Renal Cell Carcinoma, CMN = Congenital Mesoblastic Nephroma, WT = Wilms Tumor, Cor = correlation.


( A ) Clustered heatmap of viability measurements per RMS tumoroid model ( x axis ) and drug ( y axis ) , showing the Area Under the Curve ( AUC ) after treatment of the cells for 120 h with a dose - range of 0 . 1 nM to 10 µM . Low AUC ( red ) indicates high drug efficacy while high AUC ( blue ) indicates low drug efficacy . Annotated clusters of ( 1 ) MEK / ERK and γ - secretase inhibitors showing specific efficacy in RMS tumoroid models without ( RMS007 , RMS012 , RMS444 ) or low ( RMS000HQC ) fusion transcript expression , and ( 2 ) drugs that show broad efficacy across RMS tumoroid models .

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(A) Clustered heatmap of viability measurements per RMS tumoroid model (x axis) and drug (y axis), showing the Area Under the Curve (AUC) after treatment of the cells for 120 h with a dose-range of 0.1 nM to 10 µM. Low AUC (red) indicates high drug efficacy while high AUC (blue) indicates low drug efficacy. Annotated clusters of (1) MEK/ERK and γ-secretase inhibitors showing specific efficacy in RMS tumoroid models without (RMS007, RMS012, RMS444) or low (RMS000HQC) fusion transcript expression, and (2) drugs that show broad efficacy across RMS tumoroid models.


( B ) Western Blot analysis of TP53 wildtype ( WT ) and knockout ( KO ) RMS tumoroid line RMS012 . Histone 3 ( H3 ) served as loading control .

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(B) Western Blot analysis of TP53 wildtype (WT) and knockout (KO) RMS tumoroid line RMS012. Histone 3 (H3) served as loading control.


( C ) Dose - response curve of TP53 WT and KO cells treated with the Chk - 1 inhibitor prexasertib . Thin lines with numbers indicate individual biological replicates ( n = 3 ) while thick lines indicate fitted lines over all replicates . The statistical significance of the differences in fitted IC50 values between WT and KO were obtained using a two - sided t - test ( p = 0 . 008 ) .

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(C) Dose-response curve of TP53 WT and KO cells treated with the Chk-1 inhibitor prexasertib. Thin lines with numbers indicate individual biological replicates (n = 3) while thick lines indicate fitted lines over all replicates. The statistical significance of the differences in fitted IC50 values between WT and KO were obtained using a two-sided t-test (p = 0.008).


( C - J ) All panels show Drosophila 3rd instar larval eye imaginal discs with eyeless - Flippase - induced clones of cells homozygous for EMC3Δ4 labelled by the absence of ubiGFP ( green ) . The red channel shows ELAV , which labels the nuclei of photoreceptor cells . All UAS constructs were expressed under the control of GMR - GAL4 . ( C ) Expression of UAS - Rh1 ( 4C5 , in blue ) and ( D ) Na + K + ATPase ( A5 - C , in blue ) is strongly reduced in EMC3Δ4 homozygous mutant cells . ( E ) Expression of UAS - Xport - A - HA ( anti - HA , in blue ) and ( F ) UAS - fan - HA ( anti - HA , in blue ) is strongly reduced in EMC3Δ4 homozygous mutant cells . ( G ) Expression of UAS - rtv - HA ( anti - HA , in blue ) and ( H ) hid ( anti - hid , in blue ) is increased in EMC3Δ4 homozygous mutant cells . ( I - J ) Normal expression of two TA proteins in EMC3Δ4 homozygous mutant cells . ( I ) UAS - CG8814 - HA ( anti - HA , in blue ) is a TA protein containing a TMD of low hydrophobicity ( J ) UAS - Syx13 - HA is a TA protein with high hydrophobicity TMD . Scale bars represent 10 μm .

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(C-J) All panels show Drosophila 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4 labelled by the absence of ubiGFP (green). The red channel shows ELAV, which labels the nuclei of photoreceptor cells. All UAS constructs were expressed under the control of GMR-GAL4. (C) Expression of UAS-Rh1 (4C5, in blue) and (D) Na+K+ATPase (A5-C, in blue) is strongly reduced in EMC3Δ4 homozygous mutant cells. (E) Expression of UAS-Xport-A-HA (anti-HA, in blue) and (F) UAS-fan-HA (anti-HA, in blue) is strongly reduced in EMC3Δ4 homozygous mutant cells. (G) Expression of UAS-rtv-HA (anti-HA, in blue) and (H) hid (anti-hid, in blue) is increased in EMC3Δ4 homozygous mutant cells. (I-J) Normal expression of two TA proteins in EMC3Δ4 homozygous mutant cells. (I) UAS-CG8814-HA (anti-HA, in blue) is a TA protein containing a TMD of low hydrophobicity (J) UAS-Syx13-HA is a TA protein with high hydrophobicity TMD. Scale bars represent 10 μm.


( B - D ) - Immunostaining of 3rd instar larval eye imaginal discs with eyeless - Flippase - induced clones of cells homozygous for EMC3Δ4 , labelled by the absence of ubiGFP ( green ) shows reduced expression of ( B ) UAS - HA - Xport - A ( anti - HA , in blue ) but normal expression of ( C ) UAS - HA - Xport - A4L ( anti - HA , in blue ) or ( D ) UAS - Xport - A4L - HA ( anti - HA , in blue ) in EMC3 mutant homozygous cells . ELAV ( red in B and C ) marks photoreceptor cells and Na + K + ATPase ( red in D ) acts as a positive control for the presence of EMC3Δ4 homozygous mutant clones . The UAS constructs were expressed under the control of GMR - GAL4 . Scale bars represent 10 μm .

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(B-D) - Immunostaining of 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4, labelled by the absence of ubiGFP (green) shows reduced expression of (B) UAS-HA-Xport-A (anti-HA, in blue) but normal expression of (C) UAS-HA-Xport-A4L (anti-HA, in blue) or (D) UAS-Xport-A4L-HA (anti-HA, in blue) in EMC3 mutant homozygous cells. ELAV (red in B and C) marks photoreceptor cells and Na+K+ATPase (red in D) acts as a positive control for the presence of EMC3Δ4 homozygous mutant clones. The UAS constructs were expressed under the control of GMR-GAL4. Scale bars represent 10 μm.


( E - F ) Immunostaining of mosaic adult retina with eyeless - Flippase - induced clones of cells homozygous for EMC3e02662 , labelled by the absence of RFP ( red ) , show loss of ( E ) UAS - HA - Xport - A ( anti - HA , in blue ) in EMC3 mutant homozygous cells . ( F ) Expression of UAS - HA - Xport - A4L ( anti - HA , in blue ) is normal in EMC3e02662 homozygous mutant clones . The rhabdomeres are stained for actin ( Phalloidin , in green ) . Scale bars represent 10 μm .

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(E-F) Immunostaining of mosaic adult retina with eyeless-Flippase-induced clones of cells homozygous for EMC3e02662, labelled by the absence of RFP (red), show loss of (E) UAS-HA-Xport-A (anti-HA, in blue) in EMC3 mutant homozygous cells. (F) Expression of UAS-HA-Xport-A4L (anti-HA, in blue) is normal in EMC3e02662 homozygous mutant clones. The rhabdomeres are stained for actin (Phalloidin, in green). Scale bars represent 10 μm.


( B - D ) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct . " EMC5KO + EV " indicates a knockout of EMC5 stably harbouring empty vector , while ' ' EMC5KO + EMC5 " indicates EMC5KO cells rescued by inducible re - expression of a stably integrated EMC5 . ( E - G ) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct . " EMC6KO + EV " indicates a knockout of EMC6 stably harbouring empty vector , while ' ' EMC6KO + EMC6 " indicates EMC6KO cells rescued by re - expression of a stably integrated EMC6 .

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(B-D) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct. "EMC5KO+EV" indicates a knockout of EMC5 stably harbouring empty vector, while ''EMC5KO+EMC5" indicates EMC5KO cells rescued by inducible re-expression of a stably integrated EMC5. (E-G) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct. "EMC6KO+EV" indicates a knockout of EMC6 stably harbouring empty vector, while ''EMC6KO+EMC6" indicates EMC6KO cells rescued by re-expression of a stably integrated EMC6.


( C - G ) ( C - D , F ) Western - Blot of WT and EMC6KO ( 6KO ) U2OS cell lines transiently transfected with the opsin - tagged constructs probed for HA . When indicated , denatured samples were treated with PNGase to confirm glycosylation . Glycosylated proteins shift upwards in the gel and are indicated by arrows (  ) . ( E , G ) Western - Blot of WT and EMC6KO ( 6KO ) U2OS cell lines transiently transfected with the opsin - tagged constructs probed for Actin and EMC6 .

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(C-G) (C-D, F) Western-Blot of WT and EMC6KO (6KO) U2OS cell lines transiently transfected with the opsin-tagged constructs probed for HA. When indicated, denatured samples were treated with PNGase to confirm glycosylation. Glycosylated proteins shift upwards in the gel and are indicated by arrows (). (E, G) Western-Blot of WT and EMC6KO (6KO) U2OS cell lines transiently transfected with the opsin-tagged constructs probed for Actin and EMC6.


( A - B ) Immunostaining of 3rd instar larval eye imaginal discs with eyeless - Flippase - induced clones of cells homozygous for EMC3Δ4 , labelled by the absence of ubiGFP ( green ) show loss of ( A ) UAS - Xport - A - HA ( anti - HA , in blue ) and UAS - Rh1 ( 4C5 , in red ) . ( B ) Expression of UAS - Xport - A4L ( anti - HA , in blue ) and UAS - Rh1 ( 4C5 , in red ) is observed in EMC3Δ4 homozygous cells . The UAS constructs were expressed under the control of GMR - GAL4 . Scale bars represent 10 μm .

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(A-B) Immunostaining of 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4, labelled by the absence of ubiGFP (green) show loss of (A) UAS-Xport-A-HA (anti-HA, in blue) and UAS-Rh1 (4C5, in red). (B) Expression of UAS-Xport-A4L (anti-HA, in blue) and UAS-Rh1 (4C5, in red) is observed in EMC3Δ4 homozygous cells. The UAS constructs were expressed under the control of GMR-GAL4. Scale bars represent 10 μm.


( C - D ) Immunostaining of mosaic adult retinas with eyeless - Flippase - induced clones of cells homozygous for EMC3e02662 , labelled by the absence of RFP ( red ) , show loss of ( C ) TRP ( Mab83F6 , in blue ) when UAS - HA - Xport - A is expressed . ( D ) Expression of TRP ( Mab83F6 , in blue ) is observed in EMC3e02662 homozygous cells , when HA - Xport - A4L is expressed . The rhabdomeres are stained for actin ( Phalloidin , in green ) . The UAS constructs were expressed under the control of Rh1 - GAL4 . Scale bars represent 10 μm .

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(C-D) Immunostaining of mosaic adult retinas with eyeless-Flippase-induced clones of cells homozygous for EMC3e02662, labelled by the absence of RFP (red), show loss of (C) TRP (Mab83F6, in blue) when UAS-HA-Xport-A is expressed. (D) Expression of TRP (Mab83F6, in blue) is observed in EMC3e02662 homozygous cells, when HA-Xport-A4L is expressed. The rhabdomeres are stained for actin (Phalloidin, in green). The UAS constructs were expressed under the control of Rh1-GAL4. Scale bars represent 10 μm.


( E - G ) Western blot of fly heads labelled for V5 , TRP , Rh1 , Xport - A ( Xp - A ) and tubulin . ( E ) Immunoblot of fly heads expressing Rh1Gal4 ( driver ) ( left ) or Rh1 TMD1 - V5 ( right ) in Xport - A heterozygous ( Xport - A1 / TM6B ) or homozygous mutant flies ( Xport - A1 / Xport - A1 ) . Extracts were untreated ( - ) or treated ( + ) with PNGase F enzyme ( lanes 2 , 4 , 6 and 8 ) . For Rh1Gal4 , 4 fly heads were loaded per well , while for Rh1 TMD1 - V5 , 8 fly heads were loaded per well . ( F ) Immunoblot of fly heads expressing Rh1 TMD1 - 3 - V5 in Xport - A heterozygous ( Xport - A1 / TM6B ) or homozygous mutant flies ( Xport - A1 / Xport - A1 ) . Extracts were untreated ( - ) or treated ( + ) with PNGase F enzyme ( lanes 2 and 4 ) . Eight fly heads were loaded per lane . ( G ) Immunoblot of fly heads expressing Rh1 TMD1 - 5 - V5 in Xport - A heterozygote ( Xport - A1 / TM6B ) or homozygous mutant flies ( Xport - A1 / Xport - A1 ) . Extracts were untreated ( - ) or treated ( + ) with either Endo - H ( lanes 2 and 5 ) or PNGase F enzyme ( lanes 3 and 6 ) . Each lane contains approximately 2 , 5 fly heads .

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(E-G) Western blot of fly heads labelled for V5, TRP, Rh1, Xport-A (Xp-A) and tubulin. (E) Immunoblot of fly heads expressing Rh1Gal4 (driver) (left) or Rh1 TMD1-V5 (right) in Xport-A heterozygous (Xport-A1/TM6B) or homozygous mutant flies (Xport-A1/Xport-A1). Extracts were untreated (-) or treated (+) with PNGase F enzyme (lanes 2, 4, 6 and 8). For Rh1Gal4, 4 fly heads were loaded per well, while for Rh1 TMD1-V5, 8 fly heads were loaded per well. (F) Immunoblot of fly heads expressing Rh1 TMD1-3-V5 in Xport-A heterozygous (Xport-A1/TM6B) or homozygous mutant flies (Xport-A1/Xport-A1). Extracts were untreated (-) or treated (+) with PNGase F enzyme (lanes 2 and 4). Eight fly heads were loaded per lane. (G) Immunoblot of fly heads expressing Rh1 TMD1-5-V5 in Xport-A heterozygote (Xport-A1/TM6B) or homozygous mutant flies (Xport-A1/Xport-A1). Extracts were untreated (-) or treated (+) with either Endo-H (lanes 2 and 5) or PNGase F enzyme (lanes 3 and 6). Each lane contains approximately 2,5 fly heads.


. ( b ) Conjugation of LC3 and two other mammalian homologues ( GL1 and GL2 ) with phosphatidylethanolamine ( PE ) was performed as described in ref . . Complete reactions contain 1 . 5 μM Atg7 , 2 . 5 μM Atg3 , 8 - 12 μM LC3 / GL1 / GL2 , 2 mM lipid ( 30 nm liposomes composed of 10 mol % bl - PI , 55 mol % DOPE , 0 . 15 mol % Rh - DOPE and 34 . 85 mol % POPC ; see Methods for full definitions ) , 1 mM dithiothreitol and 1 mM ATP and were run at 30 ° C for 90 min . Each reaction was run on a 12 % SDS - PAGE gel and visualized by Coomassie blue stain . The mobility of the lipidated protein is faster than the corresponding non - lipidated forms . Intermediates in the reaction are also detectable , including the Atg7 - GRL1 conjugate , the Atg3 - GRL1 conjugate and a modified form of GRL1 , GRL2 and LC3 that is labelled simply as GRL1 / GRL2 / LC3 - intermediate and is probably the adenylated form of each LC3 - related protein .

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

. (b) Conjugation of LC3 and two other mammalian homologues (GL1 and GL2) with phosphatidylethanolamine (PE) was performed as described in ref. . Complete reactions contain 1.5 μM Atg7, 2.5 μM Atg3, 8-12 μM LC3/GL1/GL2, 2 mM lipid (30 nm liposomes composed of 10 mol% bl-PI, 55 mol% DOPE, 0.15 mol% Rh-DOPE and 34.85 mol% POPC; see Methods for full definitions), 1 mM dithiothreitol and 1 mM ATP and were run at 30 °C for 90 min. Each reaction was run on a 12% SDS-PAGE gel and visualized by Coomassie blue stain. The mobility of the lipidated protein is faster than the corresponding non-lipidated forms. Intermediates in the reaction are also detectable, including the Atg7-GRL1 conjugate, the Atg3-GRL1 conjugate and a modified form of GRL1, GRL2 and LC3 that is labelled simply as GRL1/GRL2/LC3-intermediate and is probably the adenylated form of each LC3-related protein.


( c ) The lipidation reactions are highly dependent on the molar percentage of DOPE in the liposomes . Reactions are as in b , but run on 400 nm liposomes . Liposomes are composed of the indicated amount of DOPE , 10 mol % bl - PI , 0 . 15 mol % Rh - DOPE and POPC .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 lipidation reactions are highly dependent on the molar percentage of DOPE in the liposomes. Reactions are as in b, but run on 400 nm liposomes. Liposomes are composed of the indicated amount of DOPE, 10 mol% bl-PI, 0.15 mol% Rh-DOPE and POPC.


( d ) Completed reactions in c were subjected to density gradient flotation to isolate only the lipidated material . Floated samples were run on SDS - PAGE and imaged by Coomassie . For c and d , the samples were prepared at the same time from the same materials and run simultaneously on multiple gels , as indicated by the vertical lines . Uncropped versions of all gels are included in Supplementary Fig . 7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Completed reactions in c were subjected to density gradient flotation to isolate only the lipidated material. Floated samples were run on SDS-PAGE and imaged by Coomassie. For c and d, the samples were prepared at the same time from the same materials and run simultaneously on multiple gels, as indicated by the vertical lines. Uncropped versions of all gels are included in Supplementary Fig. 7.


( b ) Filling the defects with inverted cone - shaped lipids inhibits lipidation . In vitro coupling reactions of GL1 were run on sonicated liposomes containing 30 mol % DOPE ( 2 mM total lipid ) as in Fig . 1 . To determine whether inverted cones influenced the reaction , increasing amounts of stearoyl - CoA were added immediately before initiating the reaction with ATP . Quantification is of n = 3 samples ; error bars represent standard deviation . P values represent a comparison with samples without stearoyl - CoA . * * P 0 . 1 ; * P 0 . 05 .

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

(b) Filling the defects with inverted cone-shaped lipids inhibits lipidation. In vitro coupling reactions of GL1 were run on sonicated liposomes containing 30 mol% DOPE (2 mM total lipid) as in Fig. 1. To determine whether inverted cones influenced the reaction, increasing amounts of stearoyl-CoA were added immediately before initiating the reaction with ATP. Quantification is of n = 3 samples; error bars represent standard deviation. P values represent a comparison with samples without stearoyl-CoA. **P 0.1; *P 0.05.


( c ) Altering the shape of PE to reduce local membrane defects inhibits the lipidation reaction . In vitro coupling reactions of GL1 were run on liposomes of three different sizes ( 400 nm and 50 nm extruded liposomes and sonicated liposomes ) and of three different lipid compositions . Lipid compositions include 30 mol % DPPE , DSPE or DOPE ( see Methods for full definitions ) , 10 % bl - PI , and POPC . Total lipid = 2 mM . Quantification is of n = 3 samples ; error bars represent standard deviation . The extent of lipidation in b and c is plotted as a percentage of total GL1 as determined by densitometry . P values represent comparison with most active liposomes ( sonicated DOPE ) . * * P 0 . 1 ; * P 0 . 05 .

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

(c) Altering the shape of PE to reduce local membrane defects inhibits the lipidation reaction. In vitro coupling reactions of GL1 were run on liposomes of three different sizes (400 nm and 50 nm extruded liposomes and sonicated liposomes) and of three different lipid compositions. Lipid compositions include 30 mol% DPPE, DSPE or DOPE (see Methods for full definitions), 10% bl-PI, and POPC. Total lipid = 2 mM. Quantification is of n = 3 samples; error bars represent standard deviation. The extent of lipidation in b and c is plotted as a percentage of total GL1 as determined by densitometry. P values represent comparison with most active liposomes (sonicated DOPE). **P 0.1; *P 0.05.


( b ) To determine the impact of curvature on lipidation , we compared liposomes with unphysiologically high surface densities of PE ( 55 mol % ) as have been used in other Atg8 or LC3 lipidation publications ( for example , refs , , ) , with liposomes having the highest densities of liposomes observed in mammalian organelle membranes ( 30 mol % ) . At physiologically relevant concentrations of liposomes ( 30 mol % ) , lipidation becomes membrane curvature dependent . In vitro coupling reactions were performed as in Fig . 1 , except that liposomes were made by extrusion through membranes with different pore diameters ( as indicated ) . Actual diameters of these liposomes were determined by dynamic light scattering ( Supplementary Table 1 ) . Top : the lipidation of GL2 , GL2 and PE proceeds efficiently and with relatively little curvature dependence on liposomes composed of 55 % PE . However , when the liposomes is reduced to 30 % , flat liposomes are no longer suitable for lipidation , while highly curved liposomes ( 100 nm diameter or less ) continue to couple effectively . Bottom : to confirm that this curvature dependence reveals the lipidated product and not the intermediate , the 30 % reactions were each run on Nycodenz flotation gradients . Only the lipidated product is recovered at the top of the gradient .

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

(b) To determine the impact of curvature on lipidation, we compared liposomes with unphysiologically high surface densities of PE (55 mol%) as have been used in other Atg8 or LC3 lipidation publications (for example, refs , , ), with liposomes having the highest densities of liposomes observed in mammalian organelle membranes (30 mol%). At physiologically relevant concentrations of liposomes (30 mol%), lipidation becomes membrane curvature dependent. In vitro coupling reactions were performed as in Fig. 1, except that liposomes were made by extrusion through membranes with different pore diameters (as indicated). Actual diameters of these liposomes were determined by dynamic light scattering (Supplementary Table 1). Top: the lipidation of GL2, GL2 and PE proceeds efficiently and with relatively little curvature dependence on liposomes composed of 55% PE. However, when the liposomes is reduced to 30%, flat liposomes are no longer suitable for lipidation, while highly curved liposomes (100 nm diameter or less) continue to couple effectively. Bottom: to confirm that this curvature dependence reveals the lipidated product and not the intermediate, the 30% reactions were each run on Nycodenz flotation gradients. Only the lipidated product is recovered at the top of the gradient.


( c ) The lipidation reaction is largely insensitive to PE concentrations when run on highly curved liposomes . Coupling of GL1 and LC3 to PE was assessed on liposomes of two sizes ( 400 nm , black squares ; sonicated , red circles ) and varying molar percentage of DOPE . The extent of lipidation is determined as in Fig . 2 . ( n = 2 for 30 and 55 % PE , n = 4 for all the other points , and error bars represent standard deviation . ) P values were determined by comparing sonicated with 400 nm samples at each PE concentration . * * P 0 . 1 ; * P 0 . 01 . Uncropped versions of all gels are included in Supplementary Fig . 7 .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 lipidation reaction is largely insensitive to PE concentrations when run on highly curved liposomes. Coupling of GL1 and LC3 to PE was assessed on liposomes of two sizes (400 nm, black squares; sonicated, red circles) and varying molar percentage of DOPE. The extent of lipidation is determined as in Fig. 2. (n = 2 for 30 and 55% PE, n = 4 for all the other points, and error bars represent standard deviation.) P values were determined by comparing sonicated with 400 nm samples at each PE concentration. **P 0.1; *P 0.01. Uncropped versions of all gels are included in Supplementary Fig. 7.


( a ) The accumulation of the Atg3 - GL1 conjugate depends on the extent of membrane curvature . In vitro GL1 coupling reactions were run as in Fig . 1 . The reactions were visualized by immunoblot against Atg3 ( top ) and by Coomassie staining of GRL1 ( bottom ) . The accumulation of Atg3 - GL1 is inversely related to the formation of the lipidated GRL1 , suggesting that the membrane - curvature - dependent step may involve Atg3 recognition of the membrane surface .

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

(a) The accumulation of the Atg3-GL1 conjugate depends on the extent of membrane curvature. In vitro GL1 coupling reactions were run as in Fig. 1. The reactions were visualized by immunoblot against Atg3 (top) and by Coomassie staining of GRL1 (bottom). The accumulation of Atg3-GL1 is inversely related to the formation of the lipidated GRL1, suggesting that the membrane-curvature-dependent step may involve Atg3 recognition of the membrane surface.


( b ) Atg3 binding to liposomes . Atg3 ( 10 μM ) was incubated at 30 ° C for 90 min with liposomes containing 0 , 30 or 55 mol % DOPE ( 5 mM lipids ) and of varying sizes ( extrusion membrane dimensions are shown on the figure ; actual final sizes were determined by dynamic light scattering ( Supplementary Table 1 ) ) . The liposome - associated Atg3 was recovered by Nycodenz density gradient centrifugation and analysed by SDS - PAGE .

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

(b) Atg3 binding to liposomes. Atg3 (10 μM) was incubated at 30 °C for 90 min with liposomes containing 0, 30 or 55 mol% DOPE (5 mM lipids) and of varying sizes (extrusion membrane dimensions are shown on the figure; actual final sizes were determined by dynamic light scattering (Supplementary Table 1)). The liposome-associated Atg3 was recovered by Nycodenz density gradient centrifugation and analysed by SDS-PAGE.


( c ) Quantification of liposome - associated Atg3 . Densitometric plot of the amount of Atg3 recovered in the top fraction in flotation assays as in b . Error bars indicate s . e . m . from three independent experiments . P values calculated as compared with putative negative control ( 0 % PE , 400 nm ) . * P 0 . 05 . Uncropped versions of all gels are included in Supplementary Fig . 7 .

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

(c) Quantification of liposome-associated Atg3. Densitometric plot of the amount of Atg3 recovered in the top fraction in flotation assays as in b. Error bars indicate s.e.m. from three independent experiments. P values calculated as compared with putative negative control (0% PE, 400 nm). *P 0.05. Uncropped versions of all gels are included in Supplementary Fig. 7.


( b ) Analysis of reaction efficiency for point mutants of the amphipathic helix . GL1 lipidation reactions were run on 400 nm and sonicated liposomes ( 30 mol % DOPE ) with the indicated Atg3 mutants . The extent of GL1 - PE formation ( top gel ) was determined from densitometry and is plotted as percentage of total GL1 ( bar graph ) . The ability to form the Atg3 - GL1 conjugate was also assessed for each mutant ( bottom immunoblot ) . Note that this conjugate forms in every case , indicating that no mutant inhibited a step upstream in the reaction . Further , this conjugate specifically accumulates in reactions where lipidation is impaired . In both panels , the samples were prepared at the same time from the same materials and run simultaneously on multiple gels , as indicated by the lines .

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

(b) Analysis of reaction efficiency for point mutants of the amphipathic helix. GL1 lipidation reactions were run on 400 nm and sonicated liposomes (30 mol% DOPE) with the indicated Atg3 mutants. The extent of GL1-PE formation (top gel) was determined from densitometry and is plotted as percentage of total GL1 (bar graph). The ability to form the Atg3-GL1 conjugate was also assessed for each mutant (bottom immunoblot). Note that this conjugate forms in every case, indicating that no mutant inhibited a step upstream in the reaction. Further, this conjugate specifically accumulates in reactions where lipidation is impaired. In both panels, the samples were prepared at the same time from the same materials and run simultaneously on multiple gels, as indicated by the lines.


( c ) Curvature dependence of K11L mutant binding to liposomes compared with wild - type Atg3 . The binding reaction was performed as in Fig . 4b on 30 DOPE % liposomes . F , float - up ; T , total input .

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

(c) Curvature dependence of K11L mutant binding to liposomes compared with wild-type Atg3. The binding reaction was performed as in Fig. 4b on 30 DOPE% liposomes. F, float-up; T, total input.


( d ) Lys 11 represents a tunable node through which curvature dependence of the lipidation reaction can be controlled . Lipidation assays were run as in b . Total GL1 lipidation was assessed from reactions with wild - type Atg3 or mutant forms of Atg3 in which position Lys 11 was changed as indicated . Lipidation efficiency on low PE / low curvature liposomes ( 30 % PE , 400 nm ) increases with increasing hydrophobicity of the new amino acid , while liposomes with high PE ( 55 % ) or high curvature ( sonicated ) are good substrates for all active forms of Atg3 . Left : Coomassie gels of individual lipidation reactions . Right : densitometry of Coomassie - stained gels revealed the ratio of GL1 - PE to the sum of all three GL1 species as in Fig . 2 . Light grey bars are sonicated liposomes ; dark grey bars are 400 nm extruded liposomes . For b , c , P values were calculated between sonicated and 400 nm samples of the same mutant . For d , P values represent comparison with WT protein on the same liposome size and composition . For all panels , n = 3 independent experiments , error bars represent standard deviation and * * P 0 . 05 ; * P 0 . 01 . Uncropped gels in Supplementary Fig . 7 .

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

(d) Lys 11 represents a tunable node through which curvature dependence of the lipidation reaction can be controlled. Lipidation assays were run as in b. Total GL1 lipidation was assessed from reactions with wild-type Atg3 or mutant forms of Atg3 in which position Lys 11 was changed as indicated. Lipidation efficiency on low PE/low curvature liposomes (30% PE, 400 nm) increases with increasing hydrophobicity of the new amino acid, while liposomes with high PE (55%) or high curvature (sonicated) are good substrates for all active forms of Atg3. Left: Coomassie gels of individual lipidation reactions. Right: densitometry of Coomassie-stained gels revealed the ratio of GL1-PE to the sum of all three GL1 species as in Fig. 2. Light grey bars are sonicated liposomes; dark grey bars are 400 nm extruded liposomes. For b,c, P values were calculated between sonicated and 400 nm samples of the same mutant. For d, P values represent comparison with WT protein on the same liposome size and composition. For all panels, n = 3 independent experiments, error bars represent standard deviation and **P 0.05; *P 0.01. Uncropped gels in Supplementary Fig. 7.


Atg3 - knockout MEFs ( Atg3 − / − ) are incapable of forming LC3 - II and do not accumulate LC3 puncta . Instead , they accumulate autophagic intermediates that are positive for Atg16 . To determine whether the amphipathic helix is critical for in vivo function , Atg3 with wild - type or mutant forms of the amphipathic helix was introduced into Atg3 − / − MEFs by lentiviral infection and rescue of the three phenotypes was tested . ( a ) Immunoblot analysis of LC3 - II , GL2 - II and GABARAP - II formation . GABARAP lipidation is significantly observed only in the presence of bafilomycin A1 ( BafA1 : 100 nM ) . Asterisk indicates a nonspecific band recognized by the Atg3 antibody .

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

Atg3-knockout MEFs (Atg3−/−) are incapable of forming LC3-II and do not accumulate LC3 puncta. Instead, they accumulate autophagic intermediates that are positive for Atg16. To determine whether the amphipathic helix is critical for in vivo function, Atg3 with wild-type or mutant forms of the amphipathic helix was introduced into Atg3−/− MEFs by lentiviral infection and rescue of the three phenotypes was tested. (a) Immunoblot analysis of LC3-II, GL2-II and GABARAP-II formation. GABARAP lipidation is significantly observed only in the presence of bafilomycin A1 (BafA1: 100 nM). Asterisk indicates a nonspecific band recognized by the Atg3 antibody.


( b ) Cells were infected as in a and transduced cells were selected with puromycin for 1 - 2 days before starvation and immunolabelling with the anti - LC3B antibody . Representative images are shown in

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

(b) Cells were infected as in a and transduced cells were selected with puromycin for 1-2 days before starvation and immunolabelling with the anti-LC3B antibody. Representative images are shown in b


( c ) The number of LC3 puncta per cell was counted manually from 22 - 96 cells per replicate . Raw data can be found in Supplementary Fig . 5 . P values represent difference from vector negative control . n = 2 - 5 independent experiments ; the precise n values and number of cells per experiment are listed in the Methods .

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

(c) The number of LC3 puncta per cell was counted manually from 22-96 cells per replicate. Raw data can be found in Supplementary Fig. 5. P values represent difference from vector negative control. n = 2-5 independent experiments; the precise n values and number of cells per experiment are listed in the Methods.


d ) Cells as in b were kept in full media before being subjected to fixation and immunolabelling with the anti - Atg16L antibody . Atg16L puncta area as a fraction of total cellular area was obtained as described in Methods . This ratio was plotted as arbitrary units . Grey dashed line indicates the value for Atg3 − / − cells rescued with wild - type Atg3 . n = 2 ( for V15K ) or 3 ( for all others ) independent experiments ; the precise n values and number of fields visualized per experiment are listed in the Methods . Raw data can be found in Supplementary Fig . 4 . Atg3 variants that support lipidation of LC3 family proteins in vitro ( Fig . 5 ) are indicated with black bars , while those that do not support lipidation are indicated with white bars . P values represent difference from wild - type rescue . * * P 0 . 1 ; * P 0 . 05 . Error bars in c , d represent s . d . for all samples of n > 2 . Uncropped versions of all gels are included in Supplementary Fig . 7 .

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

d) Cells as in b were kept in full media before being subjected to fixation and immunolabelling with the anti-Atg16L antibody. Atg16L puncta area as a fraction of total cellular area was obtained as described in Methods. This ratio was plotted as arbitrary units. Grey dashed line indicates the value for Atg3−/− cells rescued with wild-type Atg3. n = 2 (for V15K) or 3 (for all others) independent experiments; the precise n values and number of fields visualized per experiment are listed in the Methods. Raw data can be found in Supplementary Fig. 4. Atg3 variants that support lipidation of LC3 family proteins in vitro (Fig. 5) are indicated with black bars, while those that do not support lipidation are indicated with white bars. P values represent difference from wild-type rescue. **P 0.1; *P 0.05. Error bars in c,d represent s.d. for all samples of n > 2. Uncropped versions of all gels are included in Supplementary Fig. 7.


E Reproducibility of 2 independent experiments - correlation of observed changes in expression . Blue line indicates the linear fit with shaded areas as confidence intervals .

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E Reproducibility of 2 independent experiments - correlation of observed changes in expression. Blue line indicates the linear fit with shaded areas as confidence intervals.


A Number of target genes identified using differential expression for each assayed TF . * genes identified as essential for self - renewal of Hoxb8 - FL cells either in ( Basilico et al , 2020 ) .

O O O O O O O O O O O O O O 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 Number of target genes identified using differential expression for each assayed TF. *genes identified as essential for self-renewal of Hoxb8-FL cells either in (Basilico et al, 2020).