( H - K " ) IF on cross - sections of E13 . 5 optic stalks ( OS ) , and E18 . 5 and P7 optic nerves ( ONs ) with Nr2f1 ( red ) and Glast ( green , astrocytic progenitors in H - H ' ' ) , NF1A ( green , astrocytes in I - I ' ' ) or Sox10 ( green , oligodendrocyte precursors in J - K ' ' ) showing Nr2f1 expression in both astrocytic and oligodendrocytic lineages . Arrowheads point to double labeled cells . Scale bars : 50µm for mouse

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

(H-K") IF on cross-sections of E13.5 optic stalks (OS), and E18.5 and P7 optic nerves (ONs) with Nr2f1 (red) and Glast (green, astrocytic progenitors in H-H''), NF1A (green, astrocytes in I-I'') or Sox10 (green, oligodendrocyte precursors in J-K'') showing Nr2f1 expression in both astrocytic and oligodendrocytic lineages. Arrowheads point to double labeled cells. Scale bars: 50µm for mouse sections


( L - L " ) NR2F1 ( red ) and PAX6 ( green ; NR domain ) IF on sagittal sections of human eye primordia at gestational week ( GW ) 11 illustrating high NR2F1 expression in both ON and NR cells . Scale bars : 100µm for human

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L") NR2F1 (red) and PAX6 (green; NR domain) IF on sagittal sections of human eye primordia at gestational week (GW) 11 illustrating high NR2F1 expression in both ON and NR cells. Scale bars: 100µm for human sections


( M - N " ) NR2F1 ( red ) and S100β ( green , astrocytes ) IF on cross - sections of human GW11 and GW14 ONs . Higher magnifications ( M ' - N " ) show that most of S100β + astrocytes co - express NR2F1 . Scale bars : 100µm for human sections ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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") NR2F1 (red) and S100β (green, astrocytes) IF on cross-sections of human GW11 and GW14 ONs. Higher magnifications (M'-N") show that most of S100β+ astrocytes co-express NR2F1. Scale bars: 100µm for human sections,


( O - Q " ' ) NR2F1 ( red ) and SOX2 ( green in O - P ' ) or BRN3a ( green in Q - Q ' ' ' ) IF on sagittal sections of human GW14 eyes indicating high NR2F1 expression in virtually all NR progenitors ( O ' ' ' , P , P ' ) , differentiating RGCs ( Q - Q ' ' ' ) and in the majority of ON astrocytic progenitors ( O ' ) . Nuclei counterstaining ( blue ) was obtained with DAPI . Scale bars 100µm for human

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

(O-Q"') NR2F1 (red) and SOX2 (green in O-P') or BRN3a (green in Q-Q''') IF on sagittal sections of human GW14 eyes indicating high NR2F1 expression in virtually all NR progenitors (O''',P,P'), differentiating RGCs (Q-Q''') and in the majority of ON astrocytic progenitors (O'). Nuclei counterstaining (blue) was obtained with DAPI. Scale bars 100µm for human sections


( A - A ' ' ) Pax2 ( red , OD ) and TujI ( green , axons ) IF on E12 . 5 optic cup cross - sections in wild - type ( WT ) , Heterozygous ( HET ) and knock - out ( KO ) embryos showing abnormal ODs ( white dotted lines and arrowhead ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-A'') Pax2 (red, OD) and TujI (green, axons) IF on E12.5 optic cup cross-sections in wild-type (WT), Heterozygous (HET) and knock-out (KO) embryos showing abnormal ODs (white dotted lines and arrowhead).


( B - B ' ' ) Pax6 ( red , retinal progenitors ) and Tuj1 ( green ) IF indicating ectopic Pax6 + retinal tissue in HET ( B ' ) and KO embryos ( B " , arrowhead ) .

O O O O O O O O O O O O O O O O O O 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-B'') Pax6 (red, retinal progenitors) and Tuj1 (green) IF indicating ectopic Pax6+ retinal tissue in HET (B') and KO embryos (B", arrowhead).


( C - D " ) High magnification views highlight a morphological displacement of Pax2 - ( C - C " ) and Pax6 + ( D - D " ) cells . Arrowheads in ( D ' , D " ) point to ectopic Tuj1 / Pax6 + neurons .

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

(C-D") High magnification views highlight a morphological displacement of Pax2- (C-C") and Pax6+ (D-D") cells. Arrowheads in (D',D") point to ectopic Tuj1/Pax6+ neurons.


( E , E ' ) Pax2 ( red ) and Tuj1 ( green ) IF on tangential sections of E13 . 5 optic cups revealing strong OD reduction in KO embryos ( dotted line and arrowhead in E ' ) , compared to WT ( surrounded by a white dotted line in E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,E') Pax2 (red) and Tuj1 (green) IF on tangential sections of E13.5 optic cups revealing strong OD reduction in KO embryos (dotted line and arrowhead in E'), compared to WT (surrounded by a white dotted line in E).


( F ) Histogram quantifying the gradual reduction of Pax2 + OD cells in HET and KO at different ages . 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

(F) Histogram quantifying the gradual reduction of Pax2+ OD cells in HET and KO at different ages. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


( G - G ' ' ' ) Low ( G ) and high ( G ' - G ' ' ' ) magnifications of E15 . 5 optic cups stained for Pax2 ( red ) and Tuj1 ( green ) confirming Pax2 + reduction in HET and KO ODs and severe morphological malformations in KO ( arrowheads in G ' ' ' ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G''') Low (G) and high (G'-G''') magnifications of E15.5 optic cups stained for Pax2 (red) and Tuj1 (green) confirming Pax2+ reduction in HET and KO ODs and severe morphological malformations in KO (arrowheads in G''').


( H - I ' ) Tangential ( H , H ' ) and sagittal ( I , I ' ) sections of the OD in E18 . 5 WT and KO retinae stained for Brn3a ( red ; RGCs ) and Tuj1 ( green ; axons ) highlighting the malformed OD and disorganized arrangement of RGCs in KO . In ( H , H ' ) white dotted line delineate the borders of the OD and in ( I , I ' ) the RGC layer ; red dotted line surrounds mesodermal cells forming the hyaloid vessel .

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

(H-I') Tangential (H,H') and sagittal (I,I') sections of the OD in E18.5 WT and KO retinae stained for Brn3a (red; RGCs) and Tuj1 (green; axons) highlighting the malformed OD and disorganized arrangement of RGCs in KO. In (H,H') white dotted line delineate the borders of the OD and in (I,I') the RGC layer; red dotted line surrounds mesodermal cells forming the hyaloid vessel.


( J - K ' ) IF on E13 . 5 optic cup cross - sections in WT and KO embryos showing increased Pax6 ( green , retinal progenitors ) and decreased Tuj1 + ( red , differentiating cells ) in the ventral retina of HET and KO embryos . Arrowhead in ( J ' ) points to increased thickness and bending of the ventral retina , while arrowheads in ( K , K ' ) indicate Tuj1 + axons exiting the OD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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') IF on E13.5 optic cup cross-sections in WT and KO embryos showing increased Pax6 (green, retinal progenitors) and decreased Tuj1+ (red, differentiating cells) in the ventral retina of HET and KO embryos. Arrowhead in (J') points to increased thickness and bending of the ventral retina, while arrowheads in (K,K') indicate Tuj1+ axons exiting the OD.


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O 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 O 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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

(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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

(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 .

(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 :

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(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 :

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(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 )

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(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 :

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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 ) .

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

(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 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 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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR 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 :

O 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

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 .

(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).


Induction of UAS constructs and EPs by the ap promoter directed Gal4 expression within the dorsal compartment of the wing imaginal disc : ( A ) ap - Gal4 control ; ( B , E , H , J , K ) UAS - dS6K ; ( C , F , I ) UAS - S6K1dE / D3E ; ( D , E , F ) DHR3 - EP ; ( G , H , I , K ) UAS - DHR3 - RNAi and ( J ) unidirectional DHR3 - EP . The bending down of the wing indicates a slight overgrowth of the dorsal compartment , whereas a bending up reveals a slight growth deficit of this compartment .

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Induction of UAS constructs and EPs by the ap promoter directed Gal4 expression within the dorsal compartment of the wing imaginal disc: (A) ap-Gal4 control; (B, E, H, J, K) UAS-dS6K; (C, F, I) UAS-S6K1dE/D3E; (D, E, F) DHR3-EP; (G, H, I, K) UAS-DHR3-RNAi and (J) unidirectional DHR3-EP. The bending down of the wing indicates a slight overgrowth of the dorsal compartment, whereas a bending up reveals a slight growth deficit of this compartment.


Induction of UAS constructs and DHR3 - EP by the eyeless promoter - directed Gal4 expression in the developing eye : ( A ) eye - Gal4 control ; ( B , D , E ) DHR3 - EP ; ( C , D ) UAS - dTsc1 and UAS - dTsc2 ; ( E , F ) UAS - dPTEN .

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Induction of UAS constructs and DHR3-EP by the eyeless promoter-directed Gal4 expression in the developing eye: (A) eye-Gal4 control; (B, D, E) DHR3-EP; (C, D) UAS-dTsc1 and UAS-dTsc2; (E, F) UAS-dPTEN.


( B - G ) Adult wings in which the ap - Gal4 induces expression of the following UAS constructs : ( B ) ap - Gal4 control ; ( C ) UAS - dS6K ; ( D ) UAS - DHR3 - RS ; ( E ) both UAS - dS6K and UAS - DHR3 - RS ; ( F ) UAS - DHR3 - RS in a dS6Kl - 1 mutant escaper ; ( G ) UAS - dS6K , UAS - DHR3 - RS , and UAS - DHR3 - RNAi together .

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(B-G) Adult wings in which the ap-Gal4 induces expression of the following UAS constructs: (B) ap-Gal4 control; (C) UAS-dS6K; (D) UAS-DHR3-RS; (E) both UAS-dS6K and UAS-DHR3-RS; (F) UAS-DHR3-RS in a dS6Kl-1 mutant escaper; (G) UAS-dS6K, UAS-DHR3-RS, and UAS-DHR3-RNAi together.


( H ) Upper panel , H2B substrate phosphorylation by dS6K from larval protein extracts . Ubiquitous induction of UAS - DHR3 RNAi , using either a daughterless - Gal4 ( da > Ri ) or an actin - Gal4 ( ac > Ri ) driver , provoked a drop in dS6K activity , as compared with control drivers alone ( da , ac ) . Middle and lower panels are western blots detecting dS6K and α - tubulin , respectively .

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(H) Upper panel, H2B substrate phosphorylation by dS6K from larval protein extracts. Ubiquitous induction of UAS-DHR3 RNAi, using either a daughterless-Gal4 (da>Ri) or an actin-Gal4 (ac>Ri) driver, provoked a drop in dS6K activity, as compared with control drivers alone (da, ac). Middle and lower panels are western blots detecting dS6K and α-tubulin, respectively.


( A - F ) Wing imaginal discs stained with : ( A , C , D , F ) antibody to DHR3 , and ( B , E ) antibody to GFP . ( A , B ) Overexpression of DHR3 - RS and USA - GFP in the posterior compartment was induced using an engrailed - Gal4 driver . ( C ) and ( D ) ap - Gal4 drives overexpression of DHR3 - RS and DHR3 - EP , respectively . ( E , F ) Flip - out clone ( induced 3 days prior to dissection ) in a prepupal wing imaginal disc expressing UAS - DHR3 - RNAi and UAS - GFP . ( E ) Decrease in DHR3 staining in a flip - out clone labeled by GFP ( F ) , indicating that DHR3 is expressed in this tissue .

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(A-F) Wing imaginal discs stained with: (A, C, D, F) antibody to DHR3, and (B, E) antibody to GFP. (A, B) Overexpression of DHR3-RS and USA-GFP in the posterior compartment was induced using an engrailed-Gal4 driver. (C) and (D) ap-Gal4 drives overexpression of DHR3-RS and DHR3-EP, respectively. (E, F) Flip-out clone (induced 3 days prior to dissection) in a prepupal wing imaginal disc expressing UAS-DHR3-RNAi and UAS-GFP. (E) Decrease in DHR3 staining in a flip-out clone labeled by GFP (F), indicating that DHR3 is expressed in this tissue.


( G ) Western blot analysis of DHR3 in larval protein extracts from control ( Co ) and heat shock - induced UAS lines expressing DHR3 - RA ( RA ) , DHR3 - RB ( RB ) , and DHR3 - RS ( RS ) transcripts , or the DHR3 - EPs ( E1 and E2 ) .

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(G) Western blot analysis of DHR3 in larval protein extracts from control (Co) and heat shock-induced UAS lines expressing DHR3-RA (RA), DHR3-RB (RB), and DHR3-RS (RS) transcripts, or the DHR3-EPs (E1 and E2).


( H ) Western blot of the endogenous DHR3 protein in late third - instar larvae ( L3 ) , prepupae ( pp ) , and prepupae expressing an RNAi to DHR3 ( RNAipp ) . In ( G , H ) , the arrows at the left indicate the position of the DBD - containing ( high ) and DBD - lacking ( low ) DHR3 proteins .

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(H) Western blot of the endogenous DHR3 protein in late third-instar larvae (L3), prepupae (pp), and prepupae expressing an RNAi to DHR3 (RNAipp). In (G, H), the arrows at the left indicate the position of the DBD-containing (high) and DBD-lacking (low) DHR3 proteins.


( A , B ) The ap - Gal4 driver induces dS6K in combination with either ( A ) the DHR3 - EP transgene or ( B ) the EP with the EMS DHR3

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(A, B) The ap-Gal4 driver induces dS6K in combination with either (A) the DHR3-EP transgene or (B) the EP with the EMS DHR3 mutation


( D ) dS6K activity in larval protein extracts measured by H2B target phosphorylation and ( E ) corresponding western blot of dS6K in the trans - heterozygote LBD - mutant combination ( LBD ) , as compared to heterozygous larvae ( Co ) .

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(D) dS6K activity in larval protein extracts measured by H2B target phosphorylation and (E) corresponding western blot of dS6K in the trans-heterozygote LBD-mutant combination (LBD), as compared to heterozygous larvae (Co).


( F , G ) The eyeless promoter directs the flipase recombinase during eye development . Flipase allows recombination on the right arm of the second chromosome and eventually leads to an adult eye ( F ) that is homozygous for the wild - type allele of DHR3 . ( G ) Homozygosity for a DHR3K243X mutation results in a decrease in the size of the adult eye . A dotted yellow line surrounding the control eye in ( F ) has been copied and pasted on the mutant eyes ( G ) ; insets are higher magnification images of ommatidia showing misorientation of bristles .

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(F, G) The eyeless promoter directs the flipase recombinase during eye development. Flipase allows recombination on the right arm of the second chromosome and eventually leads to an adult eye (F) that is homozygous for the wild-type allele of DHR3. (G) Homozygosity for a DHR3K243X mutation results in a decrease in the size of the adult eye. A dotted yellow line surrounding the control eye in (F) has been copied and pasted on the mutant eyes (G); insets are higher magnification images of ommatidia showing misorientation of bristles.


( H ) At the scutellum , a y - marked homozygous DHR3K243X mutant bristle ( arrow ) , recognizable by its light color , is smaller than the symmetrical neighboring bristles .

(H) At the scutellum, a y-marked homozygous DHR3K243X mutant bristle (arrow), recognizable by its light color, is smaller than the symmetrical neighboring bristles.


( A ) Normalized eye size ( mm2 ) of control ( black bar ; n = 20 ) , DHR3W284X ( grey bar ; n = 18 ) and DHR3K243X ( white bar ; n = 18 ) mutants . As compared to control recombined eyes ( Co ) , DHR3W284X and DHR3K243X mutant eyes exhibit a highly significant size reduction ( * * * P < 0 . 001 ) . ( B ) Ommatidia size of control ( black bar ) , DHR3W284X ( grey bar ) , and DHR3K243X ( white bar ) mutants . As compared to control recombined eyes ( Co ) , DHR3W284X and DHR3K243X mutant ommatidia exhibit a significant size reduction ( * * P < 0 . 01 ) .

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(A) Normalized eye size (mm2) of control (black bar; n = 20), DHR3W284X (grey bar; n = 18) and DHR3K243X (white bar; n = 18) mutants. As compared to control recombined eyes (Co), DHR3W284X and DHR3K243X mutant eyes exhibit a highly significant size reduction (***P<0.001). (B) Ommatidia size of control (black bar), DHR3W284X (grey bar), and DHR3K243X (white bar) mutants. As compared to control recombined eyes (Co), DHR3W284X and DHR3K243X mutant ommatidia exhibit a significant size reduction (**P<0.01).


( C - E ) Comparison of y - marked homozygous clonal bristles ( black arrows ) to their neighboring heterozygous bristles ( white arrows ) at the wing margin . Note that control clonal bristles are of normal size and orientation ( C ) , whereas those of DHR3W284X ( D ) and DHR3K243X ( E ) are reduced in size and are misoriented . ( F ) Size measurements ( µm ) of the length of homozygous clones ( y − ) and their neighboring heterozygous ( y + ) bristles ( n = 51 for each bar ) . The size reduction of clonal versus neighbor bristles is highly significant for DHR3K243X ( * * * P < 0 . 001 ) and DHR3W284X ( * * * P < 0 . 001 ) mutants , but not for the control ( Co ) bristles ( P = 0 . 028 ) .

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(C-E) Comparison of y-marked homozygous clonal bristles (black arrows) to their neighboring heterozygous bristles (white arrows) at the wing margin. Note that control clonal bristles are of normal size and orientation (C), whereas those of DHR3W284X (D) and DHR3K243X (E) are reduced in size and are misoriented. (F) Size measurements (µm) of the length of homozygous clones (y−) and their neighboring heterozygous (y+) bristles (n = 51 for each bar). The size reduction of clonal versus neighbor bristles is highly significant for DHR3K243X (***P<0.001) and DHR3W284X (***P<0.001) mutants, but not for the control (Co) bristles (P = 0.028).


Expression of Bhlhe40 ( grey ) and Bhlhe41 ( orange ) in the indicated cell populations , data are from the Immgen Database ( microarray data set ) . CMP - common myeloid progenitors . MΦ - macrophages .

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Expression of Bhlhe40 (grey) and Bhlhe41 (orange) in the indicated cell populations, data are from the Immgen Database (microarray data set). CMP - common myeloid progenitors. MΦ - macrophages.


Flow cytometry detection of the Bhlhe40 ( grey ) and Bhlhe41 ( orange ) transcripts by the PrimeFlow RNA assay in alveolar ( right ) and peritoneal ( left ) macrophages . A probe specific for the Cd8a mRNA ( black line ) was used as a negative control . Results representative of at least four independent experiments .

Flow cytometry detection of the Bhlhe40 (grey) and Bhlhe41 (orange) transcripts by the PrimeFlow RNA assay in alveolar (right) and peritoneal (left) macrophages. A probe specific for the Cd8a mRNA (black line) was used as a negative control. Results representative of at least four independent experiments.


Cell surface phenotype and numbers of alveolar ( C ; digested lung ) and peritoneal ( D ) macrophages in WT and DKO mice . Additional gating for CD11b + cells was applied for peritoneal macrophages as indicated . Levels of CD11b expression on WT and DKO AMs are shown , and median fluorescent intensity for CD11b expression was quantified ( C , bottom left ; 3 mice per genotype ) . Absolute numbers of CD11chiCD11bloSiglecFhi alveolar macrophages from digested lungs ( C ) and CD11b + F4 / 80hi peritoneal macrophages ( D ) from WT and DKO mice are shown ( bottom ; 4 mice per genotype ) .

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Cell surface phenotype and numbers of alveolar (C; digested lung) and peritoneal (D) macrophages in WT and DKO mice. Additional gating for CD11b+ cells was applied for peritoneal macrophages as indicated. Levels of CD11b expression on WT and DKO AMs are shown, and median fluorescent intensity for CD11b expression was quantified (C, bottom left; 3 mice per genotype). Absolute numbers of CD11chiCD11bloSiglecFhi alveolar macrophages from digested lungs (C) and CD11b+F4/80hi peritoneal macrophages (D) from WT and DKO mice are shown (bottom; 4 mice per genotype).


Frequency of WT donor ( CD45 . 1 ) , DKO donor ( CD45 . 2 ) and recipient ( CD45 . 1 / 2 ) cells in the indicated BM ( LSK , CMP ) , splenic ( monocytes ) , liver ( Kupffer cells ) , peritoneal and alveolar macrophage populations of lethally irradiated recipients ≥ 6 weeks after transfer of a 1 : 1 mixture of lineage - depleted BM cells from WT and DKO mice . P values < 0 . 05 ( paired Student ' s t - test ) between LSK and other populations are shown for frequencies of WT cells . Three mixed BM chimeras analyzed ; error bars represent s . d . Representative results of three independent experiments .

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Frequency of WT donor (CD45.1), DKO donor (CD45.2) and recipient (CD45.1/2) cells in the indicated BM (LSK, CMP), splenic (monocytes), liver (Kupffer cells), peritoneal and alveolar macrophage populations of lethally irradiated recipients ≥ 6 weeks after transfer of a 1:1 mixture of lineage-depleted BM cells from WT and DKO mice. P values < 0.05 (paired Student's t-test) between LSK and other populations are shown for frequencies of WT cells. Three mixed BM chimeras analyzed; error bars represent s.d. Representative results of three independent experiments.


Representative flow - cytometric analysis of lung ( B ) macrophages as in A ( left ) . The expression of CD45 . 1 and CD45 . 2 on donor and recipient cells is shown next to the indicated gates ( right ) .

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Representative flow-cytometric analysis of lung (B) macrophages as in A (left). The expression of CD45.1 and CD45.2 on donor and recipient cells is shown next to the indicated gates (right).


Representative flow - cytometric analysis of peritoneal ( C ) macrophages as in A ( left ) . The expression of CD45 . 1 and CD45 . 2 on donor and recipient cells is shown next to the indicated gates ( right ) .

Representative flow-cytometric analysis of peritoneal (C) macrophages as in A (left). The expression of CD45.1 and CD45.2 on donor and recipient cells is shown next to the indicated gates (right).


Frequency of WT donor ( CD45 . 1 ) , DKO donor ( CD45 . 2 ) and recipient ( CD45 . 1 / 2 ) cells among the BM LSK cells , splenic monocytes and alveolar macrophages of the recipients that were irradiated with lung shielding , treated with one dose of busulfan as described in Materials and Methods , transferred with a 1 : 1 mixture of lineage - depleted BM cells from WT and DKO mice and analyzed ≥ 8 weeks after transfer . Representative flow cytometry results for alveolar macrophages ( left ) and quantification ( right ) is shown .

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Frequency of WT donor (CD45.1), DKO donor (CD45.2) and recipient (CD45.1/2) cells among the BM LSK cells, splenic monocytes and alveolar macrophages of the recipients that were irradiated with lung shielding, treated with one dose of busulfan as described in Materials and Methods, transferred with a 1:1 mixture of lineage-depleted BM cells from WT and DKO mice and analyzed ≥ 8 weeks after transfer. Representative flow cytometry results for alveolar macrophages (left) and quantification (right) is shown.


Surface phenotype of cells from the digested lungs ( right ) and quantification of donor contribution to the AM compartment ( left ) of AM - deficient Csf2rb - / - Csf2rb2 - / - recipients ( CD45 . 2 ) that were subjected to intranasal transfer of a 1 : 1 mixture of sorted fetal liver monocytes from WT ( CD45 . 1 / 2 ) and DKO ( CD45 . 2 ) E18 . 5 embryos at days 1 or 2 after birth and analyzed ≥ 7 weeks after the transfer . Cell surface phenotype of lung cells from a Csf2rb - / - Csf2rb2 - / - mouse that was not subjected to the transfer is shown for comparison .

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Surface phenotype of cells from the digested lungs (right) and quantification of donor contribution to the AM compartment (left) of AM-deficient Csf2rb-/-Csf2rb2-/-recipients (CD45.2) that were subjected to intranasal transfer of a 1:1 mixture of sorted fetal liver monocytes from WT (CD45.1/2) and DKO (CD45.2) E18.5 embryos at days 1 or 2 after birth and analyzed ≥ 7 weeks after the transfer. Cell surface phenotype of lung cells from a Csf2rb-/-Csf2rb2-/- mouse that was not subjected to the transfer is shown for comparison.


Frequency of donor - and recipient - derived AMs in WT and DKO recipients transferred at days 1 or 2 after birth intranasally with WT CD45 + cells sorted from neonatal lungs and analyzed ≥ 7 weeks after the transfer . Results for quantification ( lower panel ) are pooled from 3 independent experiments with a total of 8 WT and 9 DKO recipients ; various combinations of CD45 . 1 and CD45 . 2 were used to distinguish donor and recipient cells .

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Frequency of donor- and recipient- derived AMs in WT and DKO recipients transferred at days 1 or 2 after birth intranasally with WT CD45+ cells sorted from neonatal lungs and analyzed ≥ 7 weeks after the transfer. Results for quantification (lower panel) are pooled from 3 independent experiments with a total of 8 WT and 9 DKO recipients; various combinations of CD45.1 and CD45.2 were used to distinguish donor and recipient cells.


Mixed WT : DKO BM chimeras established as in Figure 2A were injected with EdU on three consecutive days . EdU incorporation was analyzed one day after the last injection . Steady - state WT and DKO mice were injected with EdU and analyzed as in A . Representative flow cytometry plots of EdU incorporation by AMs ( C ) and their quantification ( results pooled from two independent experiments , 7 WT and 6 DKO mice were analyzed in total ) ( B ) is shown .

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Mixed WT:DKO BM chimeras established as in Figure 2A were injected with EdU on three consecutive days. EdU incorporation was analyzed one day after the last injection. Steady-state WT and DKO mice were injected with EdU and analyzed as in A. Representative flow cytometry plots of EdU incorporation by AMs (C) and their quantification (results pooled from two independent experiments, 7 WT and 6 DKO mice were analyzed in total) (B) is shown.


WT ( CD45 . 1 ) and DKO ( CD45 . 2 ) AMs were sorted , mixed in a 1 : 1 ratio , labeled with CellTrace Violet and cultured in the presence of GM - CSF . The ratio of CD45 . 1 and CD45 . 2 cells and CellTrace Violet dilution was analyzed prior to culture ( day 0 ) and after 7 days of culture as described in Materials and Methods .

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WT (CD45.1) and DKO (CD45.2) AMs were sorted, mixed in a 1:1 ratio, labeled with CellTrace Violet and cultured in the presence of GM-CSF. The ratio of CD45.1 and CD45.2 cells and CellTrace Violet dilution was analyzed prior to culture (day 0) and after 7 days of culture as described in Materials and Methods.


RNA - seq analysis of Maf and Mafb expression in WT and DKO AMs , which were isolated by flow cytometry from mice in steady state ( top ) or from mixed BM chimeras ( bottom ) .

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RNA-seq analysis of Maf and Mafb expression in WT and DKO AMs, which were isolated by flow cytometry from mice in steady state (top) or from mixed BM chimeras (bottom).


Staining of bronchoalveolar lavage cytospins from WT and DKO mice with the lipophilic dye Oil Red O . Quantification of Oil Red O - positive cells in WT and DKO mice for individual animals . Results pooled from two independent experiments ; 4 WT and 5 DKO mice were analyzed in total .

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Staining of bronchoalveolar lavage cytospins from WT and DKO mice with the lipophilic dye Oil Red O. Quantification of Oil Red O-positive cells in WT and DKO mice for individual animals. Results pooled from two independent experiments; 4 WT and 5 DKO mice were analyzed in total.


Flow cytometric analysis of AMs from WT and DKO mice stained with the lipophilic dye BODIPY 493 / 503 . Representative results of two independent experiments with three mice per group . Analysis as in C performed with WT : DKO mixed BM chimeras . Gating on CD45 . 1 and CD45 . 2 was applied to identify cells of WT and DKO donor origin . Representative results of two independent experiments with at least three chimeras .

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Flow cytometric analysis of AMs from WT and DKO mice stained with the lipophilic dye BODIPY 493/503. Representative results of two independent experiments with three mice per group. Analysis as in C performed with WT:DKO mixed BM chimeras. Gating on CD45.1 and CD45.2 was applied to identify cells of WT and DKO donor origin. Representative results of two independent experiments with at least three chimeras.


RNA - seq analysis of Acaa1b and Hsd17b4 expression in WT and DKO AMs , which were isolated by flow cytometry from mice in steady state ( top ) or from mixed BM chimeras ( bottom ) .

RNA-seq analysis of Acaa1b and Hsd17b4 expression in WT and DKO AMs, which were isolated by flow cytometry from mice in steady state (top) or from mixed BM chimeras (bottom).


Comparison of changes in gene expression induced by Bhlhe40 / Bhlhe41 - deficiency in steady - state knockout mice ( horizontal axis ) and in mixed BM chimeras ( vertical axis ) . Log2 transformed DKO / WT fold changes are plotted . Genes with expression of > 5 TPM in at least one of the samples are displayed . Genes highlighted in magenta indicate known and putative regulators of AMs . Closed symbols - Bhlhe40 - bound genes ( Bhlhe40 ChIP - seq peak assigned to the nearest gene within a 50 kb window ) . Open symbols - genes without Bhlhe40 binding .

Comparison of changes in gene expression induced by Bhlhe40/Bhlhe41-deficiency in steady-state knockout mice (horizontal axis) and in mixed BM chimeras (vertical axis). Log2 transformed DKO/WT fold changes are plotted. Genes with expression of > 5 TPM in at least one of the samples are displayed. Genes highlighted in magenta indicate known and putative regulators of AMs. Closed symbols - Bhlhe40-bound genes (Bhlhe40 ChIP-seq peak assigned to the nearest gene within a 50 kb window). Open symbols - genes without Bhlhe40 binding.


Presence of RNA transcripts ( RNA - seq ; top ) at the Gas7 and Acaa1b genes in WT and DKO AMs ; the ChIP - seq analysis of H3K27 acetylation in WT and DKO AMs ( middle ) and Bhlhe40 binding in WT AMs ( bottom ) . All analyses performed on ex vivo AMs .

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Presence of RNA transcripts (RNA-seq; top) at the Gas7 and Acaa1b genes in WT and DKO AMs; the ChIP-seq analysis of H3K27 acetylation in WT and DKO AMs (middle) and Bhlhe40 binding in WT AMs (bottom). All analyses performed on ex vivo AMs.


Gene set enrichment analysis ( GSEA ) on dataset ranked by DKO / WT fold change in steady - state AM RNA - seq using set of genes assigned to top 1 , 000 Bhlhe40 peaks ( ranked by P value ) ( left ) or a random set of 1 , 000 genes ( right ) . Similar results were obtained with all three random sets tested . NES - normalized enrichment score . FDR False Discovery Rate .

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Gene set enrichment analysis (GSEA) on dataset ranked by DKO/WT fold change in steady-state AM RNA-seq using set of genes assigned to top 1,000 Bhlhe40 peaks (ranked by P value) (left) or a random set of 1,000 genes (right). Similar results were obtained with all three random sets tested. NES - normalized enrichment score. FDR False Discovery Rate.


Pairwise comparison of H3K27 acetylation in WT and DKO AMs performed on 2 - kb regions around the Bhlhe40 peak summits ( right - for all Bhlhe40 peaks called with P value of < 10 − 10 ; center - for top 3 , 000 Bhlhe40 peaks ( ranked by P value ) ) and around WT AM ATAC - seq peaks ( Immgen ) that did not have a Bhlhe40 peak in their close proximity ( filtered as described in Materials and Methods ) ( left ) . Volcano plots show adjusted P values ( vertical axes ) and log2 fold change ( horizontal axes ) for H3K27ac pairwise comparison in WT vs DKO AMs ; genes assigned to some of the Bhlhe40 - binding regions are labeled on the left plot .

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

Pairwise comparison of H3K27 acetylation in WT and DKO AMs performed on 2-kb regions around the Bhlhe40 peak summits (right - for all Bhlhe40 peaks called with P value of < 10−10; center - for top 3,000 Bhlhe40 peaks (ranked by P value)) and around WT AM ATAC-seq peaks (Immgen) that did not have a Bhlhe40 peak in their close proximity (filtered as described in Materials and Methods) (left). Volcano plots show adjusted P values (vertical axes) and log2 fold change (horizontal axes) for H3K27ac pairwise comparison in WT vs DKO AMs; genes assigned to some of the Bhlhe40-binding regions are labeled on the left plot.


Comparison of changes in gene expression induced by Bhlhe40 / Bhlhe41 - deficiency in steady - state knockout mice ( horizontal axis ) and in mixed BM chimeras ( vertical axis ) as shown in Fig . 5 A , but filtered for the indicated signatures ( no TPM filter applied ) . Closed symbols - genes with associated Bhlhe40 binding ( as in Fig . 5 A ) revealed by ChIP - seq . Open symbols - genes without Bhlhe40 binding . Inserts show Bhlhe40 ChIP - seq track for selected dysregulated genes .

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Comparison of changes in gene expression induced by Bhlhe40/Bhlhe41-deficiency in steady-state knockout mice (horizontal axis) and in mixed BM chimeras (vertical axis) as shown in Fig. 5 A, but filtered for the indicated signatures (no TPM filter applied). Closed symbols - genes with associated Bhlhe40 binding (as in Fig. 5 A) revealed by ChIP-seq. Open symbols - genes without Bhlhe40 binding. Inserts show Bhlhe40 ChIP-seq track for selected dysregulated genes.


Flow cytometric comparison of expression of the indicated cell - surface markers ( identified in panel A ) on WT and DKO AMs . A narrow forward / side scatter gate was applied to correct for possible differences in autofluorescence . Representative results of two independent experiments with three and four mice per group .

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Flow cytometric comparison of expression of the indicated cell-surface markers (identified in panel A) on WT and DKO AMs. A narrow forward/side scatter gate was applied to correct for possible differences in autofluorescence. Representative results of two independent experiments with three and four mice per group.


( a ) THP - 1 cells were transiently infected with BRLF1 - expressing or empty control lentivirus . Twenty - four hours later , the control or BRLF1 - expressing THP - 1 cells were primed with 40 ng / ml TPA overnight and then were either uninfected or infected with HSV - 1 ( MOI = 1 ) in serum - free medium for 12 h . The supernatants and whole cell lysates were collected and analyzed by western blotting to detect pro - caspase - 1 ( p45 ) , cleaved caspase - 1 ( p20 ) , pro - IL - 1β , mature IL - 1β , pro - IL - 18 and mature IL - 18 as indicated .

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(a) THP-1 cells were transiently infected with BRLF1-expressing or empty control lentivirus. Twenty-four hours later, the control or BRLF1-expressing THP-1 cells were primed with 40 ng/ml TPA overnight and then were either uninfected or infected with HSV-1 (MOI=1) in serum-free medium for 12 h. The supernatants and whole cell lysates were collected and analyzed by western blotting to detect pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18 and mature IL-18 as indicated.


The release of mature IL - 1β ( b ) from THP - 1 cells were measured by enzyme - linked immunosorbent assay ( ELISA ) following HSV - 1 infection .

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The release of mature IL-1β (b) from THP-1 cells were measured by enzyme-linked immunosorbent assay (ELISA) following HSV-1 infection.


The release of IL - 18 ( c ) from THP - 1 cells were measured by enzyme - linked immunosorbent assay ( ELISA ) following HSV - 1 infection .

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The release of IL-18 (c) from THP-1 cells were measured by enzyme-linked immunosorbent assay (ELISA) following HSV-1 infection.


Ramos cells were infected with wild type EBV - WT or BRLF1 - deficient EBV - ΔBRLF1 virus at a high titer ( MOI = 100 ) . After being transduced with empty control or BZLF1 - expressing lentivirus , the supernatants and cell pellets of EBV - WT - or EBV - ΔBRLF1 - harboring Ramos cells were collected after 12 h in serum - free culture , and then the levels of EBNA1 , BRLF1 , BZLF1 , pro - caspase - 1 ( p45 ) , cleaved caspase - 1 ( p20 ) , pro - IL - 1β , mature IL - 1β , pro - IL - 18 , mature IL - 18 , GSDMD and cleaved N - GSDMD in the supernatants and whole - cell lysates were analyzed as indicated ( d ) .

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Ramos cells were infected with wild type EBV-WT or BRLF1-deficient EBV-ΔBRLF1 virus at a high titer (MOI=100). After being transduced with empty control or BZLF1-expressing lentivirus, the supernatants and cell pellets of EBV-WT- or EBV-ΔBRLF1-harboring Ramos cells were collected after 12 h in serum-free culture, and then the levels of EBNA1, BRLF1, BZLF1, pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18, mature IL-18, GSDMD and cleaved N-GSDMD in the supernatants and whole-cell lysates were analyzed as indicated (d).


The release of mature IL - 1β ( e ) from Ramos cells were measured by enzyme - linked immunosorbent assay ( ELISA ) .

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The release of mature IL-1β (e) from Ramos cells were measured by enzyme-linked immunosorbent assay (ELISA).


The release of mature IL - 18 ( f ) from Ramos cells were measured by enzyme - linked immunosorbent assay ( ELISA ) .

The release of mature IL-18 (f) from Ramos cells were measured by enzyme-linked immunosorbent assay (ELISA).


( g ) After being transduced with control or BZLF1 - expressing lentivirus for 48 h , EBV - WT - and EBV - ΔBRLF1 infected Ramos cells were stained with PI and measured using fluorescent flow cytometry . Representative images of pyroptotic cells are shown and the percentages of pyroptotic cells were calculated in duplicate .

(g) After being transduced with control or BZLF1-expressing lentivirus for 48 h, EBV-WT- and EBV-ΔBRLF1 infected Ramos cells were stained with PI and measured using fluorescent flow cytometry. Representative images of pyroptotic cells are shown and the percentages of pyroptotic cells were calculated in duplicate.


( a ) GST - tagged BRLF1 - expressing plasmid or control vector was transfected into A549 cells . After HSV - 1 infection ( MOI = 1 ) for 12 h , the cells were collected , and the whole cell extracts were subjected to immunoprecipitation with GST affinity beads . The immunoprecipitated complexes were then separated by SDS - PAGE , and the image obtained from silver staining is shown .

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(a) GST-tagged BRLF1-expressing plasmid or control vector was transfected into A549 cells. After HSV-1 infection (MOI=1) for 12 h, the cells were collected, and the whole cell extracts were subjected to immunoprecipitation with GST affinity beads. The immunoprecipitated complexes were then separated by SDS-PAGE, and the image obtained from silver staining is shown.


( b ) Scramble ( sc ) or shRNAs against POLR3F and POLR3G with empty control vector ( ctr ) or BRLF1 - expressing plasmid were transfected into EBV - ΔBRLF1 - harboring HNE1 cells and A549 cells , followed by HSV - 1 infection . Thirty - six hours after the transfection of the HNE1 cells , and 12 h after HSV - 1 infection ( MOI = 1 ) of the A549 cells , the cell pellets were collected and then the cleavage of caspase - 1 and IL - 1β were measured as indicated . The release of mature IL - 1β and IL - 18 from HNE - 1 and A549 cells were measured by enzyme - linked immunosorbent assay ( ELISA ) after ultrafiltration and concentration with Amicon ® Ultra - 0 . 5 centrifugal filter devices .

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(b) Scramble (sc) or shRNAs against POLR3F and POLR3G with empty control vector (ctr) or BRLF1-expressing plasmid were transfected into EBV-ΔBRLF1-harboring HNE1 cells and A549 cells, followed by HSV-1 infection. Thirty-six hours after the transfection of the HNE1 cells, and 12 h after HSV-1 infection (MOI=1) of the A549 cells, the cell pellets were collected and then the cleavage of caspase-1 and IL-1β were measured as indicated. The release of mature IL-1β and IL-18 from HNE-1 and A549 cells were measured by enzyme-linked immunosorbent assay (ELISA) after ultrafiltration and concentration with Amicon® Ultra-0.5 centrifugal filter devices.


( c ) GFP - tagged BRLF1 wild type , BRLF1 L578A or empty vector was transfected into 293T cells with GST - tagged POLR3F or POLR3G expressing plasmids for 48 h . Cells were collected and immunoprecipitation with GST - affinity beads was performed , and then whole cell lysates and immunoprecipitated complexes were analyzed as indicated .

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(c) GFP-tagged BRLF1 wild type, BRLF1 L578A or empty vector was transfected into 293T cells with GST-tagged POLR3F or POLR3G expressing plasmids for 48 h. Cells were collected and immunoprecipitation with GST-affinity beads was performed, and then whole cell lysates and immunoprecipitated complexes were analyzed as indicated.


( d ) GFP - tagged empty vector or GFP - BRLF1 - or GFP - BRLF1 L578A - expressing plasmids were transfected into A549 cells , after which the cells were then infected with HSV - 1 , or transfected into HNE - 1 - EBV - ΔBRLF1 cells . The cells were then collected and lysed . Immunoprecipitation with GST - affinity beads was subsequently performed , and then whole - cell lysates and immunoprecipitated complexes were analyzed as indicated .

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(d) GFP-tagged empty vector or GFP-BRLF1- or GFP-BRLF1 L578A-expressing plasmids were transfected into A549 cells, after which the cells were then infected with HSV-1, or transfected into HNE-1-EBV-ΔBRLF1 cells. The cells were then collected and lysed. Immunoprecipitation with GST-affinity beads was subsequently performed, and then whole-cell lysates and immunoprecipitated complexes were analyzed as indicated.


( e ) P3HR - 1 cells were induced by TPA plus NaB into lytic infection , and the cells were then collected . Immunoprecipitation with anti - mouse IgG or BRLF1 antibody was performed , after which whole - cell lysates and immunoprecipitated complexes were analyzed as indicated .

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(e) P3HR-1 cells were induced by TPA plus NaB into lytic infection, and the cells were then collected. Immunoprecipitation with anti-mouse IgG or BRLF1 antibody was performed, after which whole-cell lysates and immunoprecipitated complexes were analyzed as indicated.


( a ) After infected with wild type EBV - WT or BRLF1 - deficient EBV - ΔBRLF1 virus at a high titer ( MOI = 100 ) , POLR3 - dependent RNA transcripts in Ramos cells were detected by RNA - seq analysis . The relative levels of cellular small RNA transcripts are shown as RNA fold changes in the heatmap in the mock ( mock group vs . mock group ) , EBV - WT ( EBV - WT group vs . mock group ) and EBV - ΔBRLF1 ( EBV - ΔBRLF1 group vs . mock group ) comparisons .

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(a) After infected with wild type EBV-WT or BRLF1-deficient EBV-ΔBRLF1 virus at a high titer (MOI=100), POLR3-dependent RNA transcripts in Ramos cells were detected by RNA-seq analysis. The relative levels of cellular small RNA transcripts are shown as RNA fold changes in the heatmap in the mock (mock group vs. mock group), EBV-WT (EBV-WT group vs. mock group) and EBV-ΔBRLF1 (EBV-ΔBRLF1 group vs. mock group) comparisons.


( b ) EBV - ΔBRLF1 - harboring HNE1 cells were transfected with control vector , BRLF1 or BRLF1 L578A in the absence or presence of BZLF1 for 48 h . The total RNAs were extracted and subjected to real - time PCR analysis .

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(b) EBV-ΔBRLF1-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A in the absence or presence of BZLF1 for 48 h. The total RNAs were extracted and subjected to real-time PCR analysis.


( c ) A549 cells were transfected with control vector , BRLF1 or BRLF1 L578A for 36 h . After the cells were infected with HSV - 1 ( MOI = 1 ) for 12 h , the total RNAs were extracted and subjected to real - time PCR analysis .

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(c) A549 cells were transfected with control vector, BRLF1 or BRLF1 L578A for 36 h. After the cells were infected with HSV-1(MOI=1) for 12 h, the total RNAs were extracted and subjected to real-time PCR analysis.


( d ) EBER1 5 ' - pppRNA detection using splint - ligation . EBV - WT - or EBV - ΔBRLF1 - harboring HNE1 cells were cotransfected with empty vector , BRLF1 or BRLF1 L578A , BRLF1 plus BZLF1 , or BRLF1 L578A plus BZLF1 for 48 h . The total RNAs were extracted , and 5 μg of each RNA was subjected to splint - ligation with FAM - labeled probe to quantify the 5 ′ - monophosphorylated EBER1 and total EBER1 RNA . The ligation products were separated using urea - PAGE , visualized by a fluorescent scanner and analyzed using ImageJ software . The ratio of 5 ' - pRNA density to total RNA density and the relative 5 ' - pppRNA level were calculated for three independent experiments .

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(d) EBER1 5'-pppRNA detection using splint-ligation. EBV-WT- or EBV-ΔBRLF1-harboring HNE1 cells were cotransfected with empty vector, BRLF1 or BRLF1 L578A, BRLF1 plus BZLF1, or BRLF1 L578A plus BZLF1 for 48 h. The total RNAs were extracted, and 5 μg of each RNA was subjected to splint-ligation with FAM-labeled probe to quantify the 5′-monophosphorylated EBER1 and total EBER1 RNA. The ligation products were separated using urea-PAGE, visualized by a fluorescent scanner and analyzed using ImageJ software. The ratio of 5'-pRNA density to total RNA density and the relative 5'-pppRNA level were calculated for three independent experiments.


( e ) EBV - WT - harboring HNE1 cells were transfected with control vector , BRLF1 or BRLF1 L578A - expressing plasmid in the presence of GST - tagged RNA polymerase subunit POLR3F for 48 h . The cells were harvested and ChIP assay was performed with anti - GST affinity beads . The POLR3F - binding promoter fragments of BSRF1 , BMLF1 , EBER1 , EBER2 , EBNA1 , 7SK , RN7SL1 and the GAPDH as negative control in the ChIP samples were detected by real - time PCR assays .

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(e) EBV-WT-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A-expressing plasmid in the presence of GST-tagged RNA polymerase subunit POLR3F for 48 h. The cells were harvested and ChIP assay was performed with anti-GST affinity beads. The POLR3F-binding promoter fragments of BSRF1, BMLF1, EBER1, EBER2, EBNA1, 7SK, RN7SL1 and the GAPDH as negative control in the ChIP samples were detected by real-time PCR assays.


( a ) Stable 293T cells in the presence of pro - caspase - 1 , pro - IL - 1β and ASC overexpression were transfected with control vector or BRLF1 with control vector , IFI16 , NLRP3 , RIG - I or AIM2 for 36 h . After the cells were infected with HSV - 1 ( MOI = 1 ) for 12 h , the cells were collected , and the cell lysates were analyzed by western blotting to determine the levels of pro - caspase - 1 , cleaved caspase - 1 , pro - IL - 1β and mature IL - 1β as indicated .

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(a) Stable 293T cells in the presence of pro-caspase-1, pro-IL-1β and ASC overexpression were transfected with control vector or BRLF1 with control vector, IFI16, NLRP3, RIG-I or AIM2 for 36 h. After the cells were infected with HSV-1 (MOI=1) for 12 h, the cells were collected, and the cell lysates were analyzed by western blotting to determine the levels of pro-caspase-1, cleaved caspase-1, pro-IL-1β and mature IL-1β as indicated.


( b ) Scramble ( sc ) or shRNAs against RIG - I and empty control vector ( ctr ) , BRLF1 - or BRLF1 L578A - expressing plasmids were co - transfected into EBV - ΔBRLF1 - harboring HNE1 cells . Thirty - six hours after the transfection , cell pellets were collected , after which the cleavage of caspase - 1 and IL - 1β was measured as indicated .

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(b) Scramble (sc) or shRNAs against RIG-I and empty control vector (ctr), BRLF1- or BRLF1 L578A-expressing plasmids were co-transfected into EBV-ΔBRLF1-harboring HNE1 cells. Thirty-six hours after the transfection, cell pellets were collected, after which the cleavage of caspase-1 and IL-1β was measured as indicated.


( c ) Analysis of ASC oligomerization . ASC - expressing plasmid was transfected into 293T cells or HNE1 cells with empty vector or GFP - BRLF1 in the presence of AIM2 - or RIG - I - expressing plasmid . Twelve hours after HSV - 1 infection ( MOI = 1 ) in 293T cells , or 48 h after EBV - ΔBRLF1 infection ( MOI = 1000 ) in HNE1 cells , the cell pellets were collected , and cell lysates were treated with DSS to induce chemical cross - linking and analyzed by western blotting for ASC oligomerization .

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(c) Analysis of ASC oligomerization. ASC-expressing plasmid was transfected into 293T cells or HNE1 cells with empty vector or GFP-BRLF1 in the presence of AIM2- or RIG-I-expressing plasmid. Twelve hours after HSV-1 infection (MOI=1) in 293T cells, or 48 h after EBV-ΔBRLF1 infection (MOI=1000) in HNE1 cells, the cell pellets were collected, and cell lysates were treated with DSS to induce chemical cross-linking and analyzed by western blotting for ASC oligomerization.


( d ) EBV - ΔBRLF1 - harboring HNE1 cells were transfected with control vector , BRLF1 or BRLF1 L578A in the absence or presence of BZLF1 for 48 h . The total RNAs were extracted and subjected to real - time PCR analysis .

(d) EBV-ΔBRLF1-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A in the absence or presence of BZLF1 for 48 h. The total RNAs were extracted and subjected to real-time PCR analysis.


( e ) A549 cells were transfected with control vector , BRLF1 or BRLF1 L578A for 36 h . After the cells were infected with HSV - 1 ( MOI = 1 ) for 12 h , the total RNAs were extracted and subjected to real - time PCR analysis .

(e) A549 cells were transfected with control vector, BRLF1 or BRLF1 L578A for 36 h. After the cells were infected with HSV-1 (MOI=1) for 12 h, the total RNAs were extracted and subjected to real-time PCR analysis.


Jurkat cells ( a ) were infected with EBV - WT or EBV - ΔBRLF1 virus ( MOI = 500 ) for 36 h . Then , the cells were collected , and the total RNAs were extracted , reverse - transcribed and analyzed by real - time PCR . Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group .

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Jurkat cells (a) were infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500) for 36 h. Then, the cells were collected, and the total RNAs were extracted, reverse-transcribed and analyzed by real-time PCR. Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group.


NK - 92MI cells ( b ) were infected with EBV - WT or EBV - ΔBRLF1 virus ( MOI = 500 ) for 36 h . Then , the cells were collected , and the total RNAs were extracted , reverse - transcribed and analyzed by real - time PCR . Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group .

NK-92MI cells (b) were infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500) for 36 h. Then, the cells were collected, and the total RNAs were extracted, reverse-transcribed and analyzed by real-time PCR. Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group.


Jurkat and NK - 92MI cells were cultured with conditional medium from EBV - WT or EBV - ΔBRLF1 - infected Ramos cells ( paracrine ) , or Jurkat and NK - 92MI cells were directly infected with EBV - WT or EBV - ΔBRLF1 viruses ( autocrine ) . ( c ) The cell pellets were collected and the whole cell extracts were analyzed by western blotting to detect TAK1 , IKKα / β and IκB phosphorylation .

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Jurkat and NK-92MI cells were cultured with conditional medium from EBV-WT or EBV-ΔBRLF1-infected Ramos cells (paracrine), or Jurkat and NK-92MI cells were directly infected with EBV-WT or EBV-ΔBRLF1 viruses (autocrine). (c) The cell pellets were collected and the whole cell extracts were analyzed by western blotting to detect TAK1, IKKα/β and IκB phosphorylation.


The cell killing activity of Jurkat cells ( d ) were measured by the detection of the LDH release from the K562 target cells .

The cell killing activity of Jurkat cells (d) were measured by the detection of the LDH release from the K562 target cells.


The cell killing activity of NK - 92MI cells ( e ) were measured by the detection of the LDH release from the K562 target cells .

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The cell killing activity of NK-92MI cells (e) were measured by the detection of the LDH release from the K562 target cells.


Anti - IL - 1β and / or anti - IL - 18 neutralizing antibodies ( NA ) , TAT - Flag or TAT - N572 peptide were added into EBV - WT - or EBV - ΔBRLF1 - infected Jurkat cells or NK - 92MI cells for 24 h . ( f ) TAK1 , IKKα and IκB phosphorylation were detected as indicated .

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Anti-IL-1β and/or anti-IL-18 neutralizing antibodies (NA), TAT-Flag or TAT-N572 peptide were added into EBV-WT- or EBV-ΔBRLF1-infected Jurkat cells or NK-92MI cells for 24 h. (f) TAK1, IKKα and IκB phosphorylation were detected as indicated.


cell killing activity of Jurkat cells ( g ) were measured .

cell killing activity of Jurkat cells (g) were measured.


cell killing activity of NK - 92MI cells ( h ) were measured .

cell killing activity of NK-92MI cells (h) were measured.


( a - d ) PBMCs were isolated and infected with EBV - WT or EBV - ΔBRLF1 virus ( MOI = 500 ) . Thirty - six hours after infection , the cells were harvested and the total RNAs were extracted , reverse - transcribed and subjected to real - time PCR analysis . The differential expression of receptors and cytokines ( a ) , cytotoxic genes ( b ) , T cell - specific genes ( c ) and NK cell - specific genes ( d ) are shown . The values are shown as the mean ± standard deviations of five analyses of PBMCs from five different health donors .

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(a-d) PBMCs were isolated and infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500). Thirty-six hours after infection, the cells were harvested and the total RNAs were extracted, reverse-transcribed and subjected to real-time PCR analysis. The differential expression of receptors and cytokines (a), cytotoxic genes (b), T cell-specific genes (c) and NK cell-specific genes (d) are shown. The values are shown as the mean ± standard deviations of five analyses of PBMCs from five different health donors.


( e - h ) FACS analysis of T and NK cell activation in PBMCs in the presence of EBV - WT or EBV - ΔBRLF1 primary infection . The PBMCs were left uninfected or infected with EBV - WT or EBV - ΔBRLF1 virus for 36 h , and the EBV - ΔBRLF1 - infected lymphocytes were incubated with additional TAT - Flag or TAT - N572 peptide for 12 h . Immunofluorescent PE - CD3 and APC - CD25 antibodies were used to detect T cell activation ( e , g ) , and PE - CD56 and APC - CD69 were used to detect NK cell activation ( f , h ) . Representative images of the FACS analysis of T cell and NK cell activation are shown ( e , f ) , and the percentages of activated T cells and NK cells were calculated and are shown as the mean ± standard deviation ( g , h ) for lymphocytes from 5 different health donors ;

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(e-h) FACS analysis of T and NK cell activation in PBMCs in the presence of EBV-WT or EBV-ΔBRLF1 primary infection. The PBMCs were left uninfected or infected with EBV-WT or EBV-ΔBRLF1 virus for 36 h, and the EBV-ΔBRLF1-infected lymphocytes were incubated with additional TAT-Flag or TAT-N572 peptide for 12 h. Immunofluorescent PE-CD3 and APC-CD25 antibodies were used to detect T cell activation (e, g), and PE-CD56 and APC-CD69 were used to detect NK cell activation (f, h). Representative images of the FACS analysis of T cell and NK cell activation are shown (e, f), and the percentages of activated T cells and NK cells were calculated and are shown as the mean ± standard deviation (g, h) for lymphocytes from 5 different health donors;


B , C Inhibitory effect of mycolactone on cell expression of CD38 ( Type II ) and CD138 , IL - 6R , and CD40 ( Type I ) transmembrane proteins ( B ) . Stimulatory effect of mycolactone on cell expression of BCMA , a Type III transmembrane protein ( C ) . Mean Fluorescence Intensities ( MFIs ) were measured in live cells 48 h after addition of mycolactone . Data are Mean MFIs ± SD from technical duplicates of 2 independent experiments , relative to vehicle - treated controls ( Ctrl ) .

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B, C Inhibitory effect of mycolactone on cell expression of CD38 (Type II) and CD138, IL-6R, and CD40 (Type I) transmembrane proteins (B). Stimulatory effect of mycolactone on cell expression of BCMA, a Type III transmembrane protein (C). Mean Fluorescence Intensities (MFIs) were measured in live cells 48 h after addition of mycolactone. Data are Mean MFIs ± SD from technical duplicates of 2 independent experiments, relative to vehicle-treated controls (Ctrl).


C Synergy between mycolactone and BZ in the MM . 1S , JIM3 and KMS - 11 cell lines , when treated as in ( B ) . Data are Mean Loewe scores ± SD shown as heatmaps . N = 6 ( cumulative data of 3 independent experiments with technical duplicates ) for MM . 1S and KMS - 11 , and N = 4 ( cumulative data of 2 independent experiments with technical duplicates ) for JIM3 . Statistical significance was assessed by Student ' s t - test : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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C Synergy between mycolactone and BZ in the MM.1S, JIM3 and KMS-11 cell lines, when treated as in (B). Data are Mean Loewe scores ± SD shown as heatmaps. N = 6 (cumulative data of 3 independent experiments with technical duplicates) for MM.1S and KMS-11, and N = 4 (cumulative data of 2 independent experiments with technical duplicates) for JIM3. Statistical significance was assessed by Student's t-test: *p<0.05; **p<0.01; ***p<0.001.


D , E MM . 1S cell were treated as in ( B ) but using carfilzomib instead of BZ ( D ) . Synergy scores ( E ) were calculated as in ( C ) . Data are Mean Loewe scores of technical duplicates , shown as heatmaps .

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D,E MM.1S cell were treated as in (B) but using carfilzomib instead of BZ (D). Synergy scores (E) were calculated as in (C). Data are Mean Loewe scores of technical duplicates, shown as heatmaps.


In MM . 1S cells treated with mycolactone and / or BZ at the indicated concentrations , or vehicle as control for 6 h : ( D ) CHOP and GADD34 mRNA levels were quantified by qPCR Data information : Shown mRNA data are Mean fold changes ( 2 - ∆ ∆ CT ) ± SD , relative to untreated controls ( cumulative data of at least 2 independent experiments with technical duplicates or triplicates ) , pairwise compared by nested one - Way ANOVA with Tukey ' s multiple comparison test , exact p - values indicated . Thapsigargin ( Tg , 2 µM , 6 h ) , Tunicamycin ( Tu , 2 µM , 6 h ) were used as positive controls .

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

In MM.1S cells treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control for 6 h: (D) CHOP and GADD34 mRNA levels were quantified by qPCR Data information: Shown mRNA data are Mean fold changes (2-∆∆CT) ± SD, relative to untreated controls (cumulative data of at least 2 independent experiments with technical duplicates or triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Thapsigargin (Tg, 2 µM, 6 h), Tunicamycin (Tu, 2 µM, 6 h) were used as positive controls.


In MM . 1S cells treated with mycolactone and / or BZ at the indicated concentrations , or vehicle as control for 6 h : ( E ) ATF6 and GAPDH protein levels in cell lysates were assessed by Western blot with molecular weight markers ( kDa ) indicated on the right ; Bands corresponding to glycosylated ( G - ATF6 ) , non - glycosylated ( NG - ATF6 ) and cleaved ATF6 are indicated by arrows ; Data information : DTT ( 4 mM , 2 h ) were used as positive controls . shown data are representative of 2 independent experiments with similar results .

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In MM.1S cells treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control for 6 h: (E) ATF6 and GAPDH protein levels in cell lysates were assessed by Western blot with molecular weight markers (kDa) indicated on the right; Bands corresponding to glycosylated (G-ATF6), non-glycosylated (NG-ATF6) and cleaved ATF6 are indicated by arrows; Data information: DTT (4 mM, 2 h) were used as positive controls. shown data are representative of 2 independent experiments with similar results.