Figure 3 ( A - D ) Ki67 ( green ; mitotically active progenitors ) and Tuj1 ( red ; post - mitotic neurons ) IF at E14 . 5 ( A , A ' ) , E16 . 5 ( B , B ' ) and E18 . 5 ( C , C ' ) of lateral pallia of wild - type ( WT ) versus mutant ( KO ) brains . Ki67 + proliferating cells ( quantified in D ) accumulate in mutant cortices . n ≥ 3 brains . Data information : Nuclei ( blue ) were stained with DAPI . the number of positive cells was quantified in 100µm - width boxes , randomly placed across the LP . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; SVZ : subventricular zone ; VZ : ventricular zone . ( E - F ' ) Tbr2 ( green ; IPs ) and Pax6 ( red ; radial glia cells ) IF of E13 . 5 ( E , E ' ) and E17 . 5 ( F , F ' ) lateral pallia of WT and KO brains . Data information : Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; SVZ : subventricular zone ; VZ : ventricular zone . Quantification of different classes of cortical progenitors : apical radial glia cells ( aRGs ) as Pax6 + NPs in VZ ( G ) , basal intermediate progenitors ( IPs ) as Tbr2 + cells in SVZ ( H ) n ≥ 3 brains . Data information : the number of positive cells was quantified in 100µm - width boxes , randomly placed across the LP . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Quantification of different classes of cortical progenitors : , basal RGs as Pax6 + cells in outer SVZ ( I ) . n ≥ 3 brains . Data information : the number of positive cells was quantified in 100µm - width boxes , randomly placed across the LP . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( J - L ) E12 . 5 dorsal brain views ( J , J ' ) and representative sections of posterior hemispheres ( K , K ' ) , showing a vesicle enlargement in mutant brains ( K ' ) . The extension of the ventricular surface was quantified along the A - P axis ( L ) ; red color code represents Nr2f1 gradient in WT brains . 12µm - thick sections were collected on series of 10 slides ; consecutive measurement levels are 120µm apart from each other . n ≥ 3 brains . Data information : In graphs , data are represented as means ± SEM . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( N - P ) GFP ( green ; expressed under Sox2 promoter ) and RFP ( red ; expressed under Tis21 promoter ) IF of E13 . 5 lateral pallia , 18 - hours after IUE . The proportion of single or double positive NPs is shown in pie charts in ( P ) . n = 3 electroporated brains . Data information : Nuclei ( blue ) were stained with DAPI . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( Q - R ' ) Ki67 ( green ; progenitors ) and EdU ( red ) IF of E13 . 5 WT ( Q , Q ' ) and KO ( R , R ' ) embryos , injected with EdU at E12 . 5 . Differentiating cells ( EdU + Ki67 - ) are located in the IZ / CP ; percentages shown in ( Q ' , R ' ) were obtained from n ≥ 4 sections of n ≥ 2 brains . Data information : Nuclei ( blue ) were stained with DAPI . Student t - test ( Q ' , R ' ; E13 . 5 , EdU injected at E12 . 5 ; WT / KO : * = 0 . 03967 ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; SVZ : subventricular zone ; VZ : ventricular zone .

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

Figure 3(A-D) Ki67 (green; mitotically active progenitors) and Tuj1 (red; post-mitotic neurons) IF at E14.5 (A,A'), E16.5 (B,B') and E18.5 (C,C') of lateral pallia of wild-type (WT) versus mutant (KO) brains. Ki67+ proliferating cells (quantified in D) accumulate in mutant cortices. n ≥ 3 brains. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.(E-F') Tbr2 (green; IPs) and Pax6 (red; radial glia cells) IF of E13.5 (E,E') and E17.5 (F,F') lateral pallia of WT and KO brains. Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.Quantification of different classes of cortical progenitors: apical radial glia cells (aRGs) as Pax6+ NPs in VZ (G), basal intermediate progenitors (IPs) as Tbr2+ cells in SVZ (H) n ≥ 3 brains. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).Quantification of different classes of cortical progenitors: , basal RGs as Pax6+ cells in outer SVZ (I). n ≥ 3 brains. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).(J-L) E12.5 dorsal brain views (J,J') and representative sections of posterior hemispheres (K,K'), showing a vesicle enlargement in mutant brains (K'). The extension of the ventricular surface was quantified along the A-P axis (L); red color code represents Nr2f1 gradient in WT brains. 12µm-thick sections were collected on series of 10 slides; consecutive measurement levels are 120µm apart from each other. n ≥ 3 brains. Data information: In graphs, data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).(N-P) GFP (green; expressed under Sox2 promoter) and RFP (red; expressed under Tis21 promoter) IF of E13.5 lateral pallia, 18-hours after IUE. The proportion of single or double positive NPs is shown in pie charts in (P). n = 3 electroporated brains. Data information: Nuclei (blue) were stained with DAPI. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).(Q-R') Ki67 (green; progenitors) and EdU (red) IF of E13.5 WT (Q,Q') and KO (R,R') embryos, injected with EdU at E12.5. Differentiating cells (EdU+Ki67-) are located in the IZ/CP; percentages shown in (Q',R') were obtained from n ≥ 4 sections of n ≥ 2 brains. Data information: Nuclei (blue) were stained with DAPI. Student t-test (Q',R'; E13.5, EdU injected at E12.5; WT/KO: *=0.03967 (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.


Figure 4 ( A - D ) Graphs showing the average number of PH3 + mitotic figures in the posterior lateral pallium of WT and mutant cortices . The total number of mitoses ( A ) as well as the number of dividing cells in VZ ( Pax6 + Sox2 + aRGs ; B ) , SVZ ( Tbr2 + IPs ; C ) and outer ( basal - most ) region of SVZ ( oSVZ / IZ ; Pax6 + oRGs ; D ) are shown . For graphs ( B - D ) , statistical analysis by 2 - way ANOVA n ≥ 3 brains per age / genotype . Data information : the number of positive cells was quantified in 100µm - width boxes , randomly placed across the lateral pallium . In graphs , data are represented as means ± SEM . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . SVZ : subventricular zone ; VZ : ventricular zone . ( E ) Cell cycle duration at different embryonic ages ( from E10 . 5 to E16 . 5 ) in WT ( blue bars ) or KO embryos ( orange line ) , as quantified upon a double EdU / BrdU injection protocol in posterior - most LP n ≥ 3 brains per age / genotype . Data information : the number of positive cells was quantified in 100µm - width boxes , randomly placed across the lateral pallium . In graphs , data are represented as means ± SEM . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( F - G ' ) Ki67 ( green ; NPs ) and EdU ( red ) IF in WT ( F , F ' ) and KO ( G , G ' ) after 6 hours consecutive EdU incorporation . Note the higher number of labelled NPs in KO cortices ( G ' ) suggesting faster cell cycle progression and / or longer S - phase . Data information : Nuclei ( blue ) were stained with DAPI . ( H ) Best linear fit of EdU + Ki67 + NP percentage after 8 hours EdU cumulative labelling in WT ( blue line ) and KO ( orange line ) brains . The x - and y - intercept are proportional to the S - phase length and to the number of cycling cells , respectively , while the steepness of the line is proportional to cell cycle duration . WT : y = 11 . 66x + 24 . 62 ; KO : y = 13 . 54x + 32 . 96 ) . n ≥ 2 brains per time point / genotype . Data information : the number of positive cells was quantified in 100µm - width boxes , randomly placed across the lateral pallium . In graphs , data are represented as means ± SEM . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( I - K ) PH3 ( green ; dividing NPs ) and Pax6 ( red ; aRGs ) IF of E13 . 5 WT ( I ) and KO ( J ) lateral pallia . Strong and weak / punctate PH3 patterns show M - phase and G2 - phase cells , respectively . G2 - phase NP percentage is quantified in ( K ) . As a result of shorter G1 - phase and globally shorter cell cycle time , the percentage of G2 - phase cells is increased in the KO NP pool . n ≥ 2 brains . Data information : Nuclei ( blue ) were stained with DAPI . the number of positive cells was quantified in 100µm - width boxes , randomly placed across the lateral pallium . In graphs , data are represented as means ± SEM . Student t - test ( K ; * P < 0 . 05 ) Scale bars : 50µm . SVZ : subventricular zone ; VZ : ventricular zone . ( L ) Cell cycle time of E13 . 5 NPs in the lateral pallia of WT ( upper column ) and KO cells ( lower column ) , as calculated by EdU cumulative labelling and PH3 staining KO NPs have a 16 . 1 % faster cell cycle time , due to a 30 . 9 % shorter G1 - phase compensating a 14 . 3 % longer S - phase . n ≥ 3 brains . In graphs , data are represented as means ± SEM . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

Figure 4(A-D) Graphs showing the average number of PH3+ mitotic figures in the posterior lateral pallium of WT and mutant cortices. The total number of mitoses (A) as well as the number of dividing cells in VZ (Pax6+Sox2+ aRGs; B), SVZ (Tbr2+ IPs; C) and outer (basal-most) region of SVZ (oSVZ/IZ; Pax6+ oRGs; D) are shown. For graphs (B-D), statistical analysis by 2-way ANOVA n ≥ 3 brains per age/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001). SVZ: subventricular zone; VZ: ventricular zone.(E) Cell cycle duration at different embryonic ages (from E10.5 to E16.5) in WT (blue bars) or KO embryos (orange line), as quantified upon a double EdU/BrdU injection protocol in posterior-most LP n ≥ 3 brains per age/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).(F-G') Ki67 (green; NPs) and EdU (red) IF in WT (F,F') and KO (G,G') after 6 hours consecutive EdU incorporation. Note the higher number of labelled NPs in KO cortices (G') suggesting faster cell cycle progression and/or longer S-phase. Data information: Nuclei (blue) were stained with DAPI.(H) Best linear fit of EdU+Ki67+ NP percentage after 8 hours EdU cumulative labelling in WT (blue line) and KO (orange line) brains. The x- and y-intercept are proportional to the S-phase length and to the number of cycling cells, respectively, while the steepness of the line is proportional to cell cycle duration. WT: y = 11.66x +24.62; KO: y = 13.54x +32.96). n ≥ 2 brains per time point/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).(I-K) PH3 (green; dividing NPs) and Pax6 (red; aRGs) IF of E13.5 WT (I) and KO (J) lateral pallia. Strong and weak/punctate PH3 patterns show M-phase and G2-phase cells, respectively. G2-phase NP percentage is quantified in (K). As a result of shorter G1-phase and globally shorter cell cycle time, the percentage of G2-phase cells is increased in the KO NP pool. n ≥ 2 brains. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. Student t-test (K; *P<0.05) Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.(L) Cell cycle time of E13.5 NPs in the lateral pallia of WT (upper column) and KO cells (lower column), as calculated by EdU cumulative labelling and PH3 staining KO NPs have a 16.1% faster cell cycle time, due to a 30.9% shorter G1-phase compensating a 14.3% longer S-phase. n ≥ 3 brains. In graphs, data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


Figure 5 ( A - B ' ) Pax6 ( green ; aRGs ) and Nr2f1 ( red ) IF of E12 . 5 WT ( A , A ' ) and KO ( B , B ' ) lateral pallia . Data information : nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . SVZ : subventricular zone ; VZ : ventricular zone . ( C ) Pax6 and Nr2f1 pixel intensity quantification at E12 . 5 and E14 . 5 indicating increased Pax6 levels upon Nr2f1 removal . n ≥ 3 brains . Data information : the pixel intensity was quantified in 100µm - width boxes , randomly placed across the lateral pallium . Data are represented as means ± SEM . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( D ) Real time RT - PCR quantification of Nr2f1 and Pax6 expression in E12 . 5 cortices . n ≥ 3 cortices . Data information : Data are represented as means ± SEM . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( E - G ' ' ) GFP ( green ; electroporated cells ) , Pax6 ( red ) and Nr2f1 ( blue ) IF of pCIG2 - Nr2f1 - IRES - GFP electroporated brains at E13 . 5 ( 24 - hours after IUE ) . VZ ( F - F ' ' ) and CP ( G - G ' ' ) regions are shown at high magnification . Arrowheads point to GFP + electroporated cells over - expressing Nr2f1 ( blue in F ' , G ' ) , down - regulating Pax6 ( F ' ' ) and rapidly migrating out of the VZ ( G - G ' ' ) . Scale bars : 50µm . SVZ : subventricular zone ; VZ : ventricular zone . ( H ) Scatter plot showing Pax6 and Nr2f1 pixel intensity of VZ electroporated progenitors , comparing Nr2f1 overexpressing cells ( orange dots ) with control pCIG2 - GFP electroporated cells ( blue dots ) . Average Nr2f1 and Pax6 pixel intensities of the 2 populations were compared by 2 - way ANOVA and resulted significantly different ( * * * = < 0 . 0001 ) . n = 2 electroporated brains . Data information Data are represented as means ± SEM . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( I ) Real time RT - PCR of cell cycle genes comparing WT ( blue line ) and KO ( orange line ) cortices . CyclinD1 and P21 transcripts are down - regulated whereas Dct is up - regulated in mutants . n = 3 cortices . Data information : Data are represented as means ± SEM . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( J - L ) GFP ( green ; electroporated cells ) and P21 ( red ) IF of E14 . 5 cortex electroporated 48 hours earlier with control PX458 plasmid ( J ) or CRISPR / Cas9 - expressing plasmid directed against Nr2f1 sequence ( PX458 - αNr2f1 ; K ) . Percentage of P21 / GFP double positive cells ( L ) . Arrowheads in ( J , K ) point to P21 + cells . n = 2 electroporated brains . Data information , nuclei ( blue ) were stained with DAPI . Student t - test ( L ; * P < 0 . 05 ( N ) Graph showing the number of differentially expressed genes ( DEG up - regulated ( UP ) or down - regulated ( DOWN ) in KO compared to WT , as detected by RNA - Seq of E15 . 5 neocortices . Data information : For statistical analysis of RNA - Seq data , see Methods . ( P ) Hierarchical clustering and heatmap of the expression level of DEGs belonging to the " neuron differentiation " Heat map color scale indicates normalized gene expression from high ( red ) to low ( white ) level . Data information : For statistical analysis of RNA - Seq data see Methods .

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

Figure 5(A-B') Pax6 (green; aRGs) and Nr2f1 (red) IF of E12.5 WT (A,A') and KO (B,B') lateral pallia. Data information: nuclei (blue) were stained with DAPI. Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.(C) Pax6 and Nr2f1 pixel intensity quantification at E12.5 and E14.5 indicating increased Pax6 levels upon Nr2f1 removal. n ≥ 3 brains. Data information: the pixel intensity was quantified in 100µm-width boxes, randomly placed across the lateral pallium. Data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).(D) Real time RT-PCR quantification of Nr2f1 and Pax6 expression in E12.5 cortices. n ≥ 3 cortices. Data information: Data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).(E-G'') GFP (green; electroporated cells), Pax6 (red) and Nr2f1 (blue) IF of pCIG2-Nr2f1-IRES-GFP electroporated brains at E13.5 (24-hours after IUE). VZ (F-F'') and CP (G-G'') regions are shown at high magnification. Arrowheads point to GFP+ electroporated cells over-expressing Nr2f1 (blue in F',G'), down-regulating Pax6 (F'') and rapidly migrating out of the VZ (G-G''). Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.(H) Scatter plot showing Pax6 and Nr2f1 pixel intensity of VZ electroporated progenitors, comparing Nr2f1 overexpressing cells (orange dots) with control pCIG2-GFP electroporated cells (blue dots). Average Nr2f1 and Pax6 pixel intensities of the 2 populations were compared by 2-way ANOVA and resulted significantly different (***= <0.0001). n = 2 electroporated brains. Data information Data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).(I) Real time RT-PCR of cell cycle genes comparing WT (blue line) and KO (orange line) cortices. CyclinD1 and P21 transcripts are down-regulated whereas Dct is up-regulated in mutants. n = 3 cortices. Data information: Data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).(J-L) GFP (green; electroporated cells) and P21 (red) IF of E14.5 cortex electroporated 48 hours earlier with control PX458 plasmid (J) or CRISPR/Cas9-expressing plasmid directed against Nr2f1 sequence (PX458-αNr2f1; K). Percentage of P21/GFP double positive cells (L). Arrowheads in (J,K) point to P21+ cells. n = 2 electroporated brains. Data information , nuclei (blue) were stained with DAPI. Student t-test (L; *P<0.05(N) Graph showing the number of differentially expressed genes (DEG up-regulated (UP) or down-regulated (DOWN) in KO compared to WT, as detected by RNA-Seq of E15.5 neocortices. Data information: For statistical analysis of RNA-Seq data , see Methods.(P) Hierarchical clustering and heatmap of the expression level of DEGs belonging to the "neuron differentiation" Heat map color scale indicates normalized gene expression from high (red) to low (white) level. Data information: For statistical analysis of RNA-Seq data see Methods.


Figure 6 ( A ) Real time RT - PCR analysis of Pax6 expression in neurospheres with different genotypes , as indicated . n = 3 cortices . N KO P HET : Nr2f1 KO , Pax6 HET . Data information : Data are represented as means ± SEM . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( B ) Representative images of Nr2f1 KO Pax6 HET neurospheres obtained from E15 . 5 neocortices and cultured in vitro . Scale bars : 50µm . ( C ) Graph showing the number of neurospheres per P100 cell plate , at different steps , as indicated . While Nr2f1 KO neurospheres ( orange line ) proliferate for a longer time compared to WT ones ( blue line ) , the loss of one Pax6 allele ( gray line ) almost restores normal proliferation rate and exhaustion time . Complete Pax6 loss is not compatible with stem cell renewal ( yellow line ) . n ≥ 3 culture wells from n = 2 independent batches . Data information : Data are represented as means ± SEM . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( D - E ) Paired cell analysis after neurosphere dissociation and 24 - hour culture at clonal density . Couples of dividing cells were identified by Map2 ( red ; N , neurons ) and BLBP ( green ; P , progenitors ) IF . Pie charts in ( E ) show the proportion of P - P ( proliferative ; D ) , P - N ( asymmetric differentiative ; D ' ) or N - N ( symmetric differentiative ; D ' ' ) couples in WT , Nr2f1KO and Nr2f1KO Pax6HET animals . n ≥ 3 samples from n = 2 culture batches . Data information : Nuclei ( blue ) were stained with DAPI . Data are represented as means ± SEM . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 Scale bars : 50µm . ( F - I ) Pax6 ( green ) and Nr2f1 ( red ) IF in the posterior - most region of E12 . 5 cortex , showing Pax6 upregulation in Nr2f1 KO animals ( G , G ' ) compared to WT ( F ' , F ' ' ) . Normal Pax6 levels can be restored by loss of one Pax6 allele ( Nr2f1 KO , Pax6 HET ; H , H ' ) . n ≥ 3 brains . N KO : Nr2f1 KO ; P HET : Pax6 HET . Data information : Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . Pixel intensity quantification of Nr2f1 ( blue ) and Pax6 ( orange ) is shown in ( I ) . n ≥ 3 brains . N KO : Nr2f1 KO ; P HET : Pax6 HET . Data information : the pixel intensity or the number of positive cells was quantified in 100µm - width boxes , randomly placed across the LP . Data are represented as means ± SEM . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( J ) Cell cycle duration at E12 . 5 in different mutants as indicated , quantified in posterior regions with double EdU / BrdU injection protocol n ≥ 6 sections from n = 2 brains . Data information : the pixel intensity or the number of positive cells was quantified in 100µm - width boxes , randomly placed across the LP . Data are represented as means ± SEM . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( K ) Insets showing representative γ - Tubulin ( green ) and Phospho - Vimentin ( red ) IF of the cortical VZ surface to evaluate the orientation of the cleavage plane of dividing aRGs . Pie charts show the percentage of vertical ( green ) , oblique ( yellow ) and horizontal ( red ) division planes of mitotic figures in the E12 . 5 lateral pallium of animals with different genotypes , as indicated . n ≥ 2 brains per genotype . Data information : Nuclei ( blue ) were stained with DAPI . Data are represented as means ± SEM . 2 - way ANOVA ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . cale bars : 50µm .

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

Figure 6(A) Real time RT-PCR analysis of Pax6 expression in neurospheres with different genotypes, as indicated. n = 3 cortices. N KO P HET: Nr2f1 KO, Pax6 HET. Data information: Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).(B) Representative images of Nr2f1 KO Pax6 HET neurospheres obtained from E15.5 neocortices and cultured in vitro. Scale bars: 50µm.(C) Graph showing the number of neurospheres per P100 cell plate, at different steps, as indicated. While Nr2f1 KO neurospheres (orange line) proliferate for a longer time compared to WT ones (blue line), the loss of one Pax6 allele (gray line) almost restores normal proliferation rate and exhaustion time. Complete Pax6 loss is not compatible with stem cell renewal (yellow line). n ≥ 3 culture wells from n = 2 independent batches. Data information: Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).(D-E) Paired cell analysis after neurosphere dissociation and 24-hour culture at clonal density. Couples of dividing cells were identified by Map2 (red; N, neurons) and BLBP (green; P, progenitors) IF. Pie charts in (E) show the proportion of P-P (proliferative; D), P-N (asymmetric differentiative; D') or N-N (symmetric differentiative; D'') couples in WT, Nr2f1KO and Nr2f1KO Pax6HET animals. n ≥ 3 samples from n = 2 culture batches. Data information: Nuclei (blue) were stained with DAPI. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001 Scale bars: 50µm.(F-I) Pax6 (green) and Nr2f1 (red) IF in the posterior-most region of E12.5 cortex, showing Pax6 upregulation in Nr2f1 KO animals (G,G') compared to WT (F',F''). Normal Pax6 levels can be restored by loss of one Pax6 allele (Nr2f1 KO, Pax6 HET; H,H'). n ≥ 3 brains. N KO: Nr2f1 KO; P HET: Pax6 HET. Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm.Pixel intensity quantification of Nr2f1 (blue) and Pax6 (orange) is shown in (I). n ≥ 3 brains. N KO: Nr2f1 KO; P HET: Pax6 HET. Data information: the pixel intensity or the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).(J) Cell cycle duration at E12.5 in different mutants as indicated, quantified in posterior regions with double EdU/BrdU injection protocol n ≥ 6 sections from n = 2 brains. Data information: the pixel intensity or the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).(K) Insets showing representative γ-Tubulin (green) and Phospho-Vimentin (red) IF of the cortical VZ surface to evaluate the orientation of the cleavage plane of dividing aRGs. Pie charts show the percentage of vertical (green), oblique (yellow) and horizontal (red) division planes of mitotic figures in the E12.5 lateral pallium of animals with different genotypes, as indicated. n ≥ 2 brains per genotype. Data information: Nuclei (blue) were stained with DAPI. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001). cale bars: 50µm.


Figure 7 ( A - C ) NR2F1 expression in cerebral organoids during neural induction and differentiation in vitro ( day 40 and day 70 , respectively ) , together with the NP markers PAX6 ( green in A ) , SOX2 ( green in B , B ' ) and DCX as a neural marker ( blue in A , B , B ' ' ) . Quantification of NR2F1 levels in NPs is shown in ( C ) . n ≥ 4 organoids from n = 2 batches . Data information : the pixel intensity or the number of positive cells were quantified in 100µm - width boxes , randomly placed across the cortex / organoid neuroepithelia In graphs , data are represented as means ± SEM . 2 - way ANOVA test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . ( D - G ) NR2F1 ( red ) and PAX6 ( green ) IF in day 40 organoids . Neuroepithelia with high NR2F1 levels show weak PAX6 expression ( E , E ' ) , and vice versa ( F , F ' ) . Quantification by pixel intensity analysis ( G ) . n ≥ 4 organoids from n = 2 batches . Data information : Nuclei ( blue stained with DAPI . the pixel intensity or the number of positive cells were quantified in 100µm - width boxes , randomly placed across the cortex / organoid neuroepithelia In graphs , data are represented as means ± SEM . 2 - way ANOVA test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . ( I - J ) NR2F1 ( red ) and GFP ( green ) IF in day 40 human brain organoids upon electroporation of the PX458 - αNR2F1 plasmid . In ( I , I ' ) , GFP + NR2F1 - cells ( empty arrowheads ) and a GFP + NR2F1 + cell ( white arrowhead ) are shown ; quantification in ( J ) . n ≥ 6 organoids from n = 2 batches . Data information : Nuclei ( blue stained with DAPI . the pixel intensity or the number of positive cells were quantified in 100µm - width boxes , randomly placed across the cortex / organoid neuroepithelia In graphs , data are represented as means ± SEM . 2 - way ANOVA test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . ( K - M ) TUJ1 ( red ; differentiating neurons ) and GFP ( green ) IF 7 days after electroporation of control ( K ) and PX458 - αNr2f1 plasmids ( L ) . Co - expression of GFP with SOX2 or TUJ1 distinguishes neural progenitors ( NPs ) from neurons ( Ns ) , as quantified in ( M ) . Arrowheads in ( K , L ) point to TUJ1 + GFP + cells . n ≥ 6 organoids from n = 2 batches . Data information : Nuclei ( blue stained with DAPI . the number of positive cells was normalized over the total number of GFP + cells . In graphs , data are represented as means ± SEM . 2 - way ANOVA test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . ( N ) Red and green arrowheads in ( N ) point to a SOX2 + ( red ) progenitor and a TUJ1 + ( blue ) neuron , respectively . Scale bars : 50µm .

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

Figure 7(A-C) NR2F1 expression in cerebral organoids during neural induction and differentiation in vitro (day 40 and day 70, respectively), together with the NP markers PAX6 (green in A), SOX2 (green in B,B') and DCX as a neural marker (blue in A,B,B''). Quantification of NR2F1 levels in NPs is shown in (C). n ≥ 4 organoids from n = 2 batches. Data information: the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.(D-G) NR2F1 (red) and PAX6 (green) IF in day 40 organoids. Neuroepithelia with high NR2F1 levels show weak PAX6 expression (E,E'), and vice versa (F,F'). Quantification by pixel intensity analysis (G). n ≥ 4 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.(I-J) NR2F1 (red) and GFP (green) IF in day 40 human brain organoids upon electroporation of the PX458-αNR2F1 plasmid. In (I,I'), GFP+NR2F1- cells (empty arrowheads) and a GFP+NR2F1+ cell (white arrowhead) are shown; quantification in (J). n ≥ 6 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.(K-M) TUJ1 (red; differentiating neurons) and GFP (green) IF 7 days after electroporation of control (K) and PX458-αNr2f1 plasmids (L). Co-expression of GFP with SOX2 or TUJ1 distinguishes neural progenitors (NPs) from neurons (Ns), as quantified in (M). Arrowheads in (K,L) point to TUJ1+ GFP+ cells. n ≥ 6 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the number of positive cells was normalized over the total number of GFP+ cells. In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.(N) Red and green arrowheads in (N) point to a SOX2+ (red) progenitor and a TUJ1+ (blue) neuron, respectively. Scale bars: 50µm.


Figure 2Relative expression and co - localization of IL6 mRNA and IL - 6 Signaling Scoring in integrated skin cells by blended UMAPs . Quantification by real - time qPCR analysis of Il6 mRNA in the skin of naïve and irradiated ( 15Gy ) WT mice at indicated times , ( n = 3 - 6 ) . Data information : Data represent mean ± SD ( B , * P < 0 . 05 , * * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 001 by one - way ANOVA with Dunnet ' s multiple comparisons test ( B ) , five independent experiments . Quantification of ventral hair loss area 21 days post - IR ( 15Gy ) in WT and IL - 6 - / - mice , ( n = 11 ) . Photographic images showing hair loss ( area demarked by yellow dashed line ) 21 days post - IR ( 15 Gy ) in wild type ( WT ) and IL - 6 - / - mice . Data information : Data represent mean ± SD C , * P < 0 . 05 , * * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 001 by , two - tailed Mann - Whitney test ( C , ; five independent experiments . Histochemical ( H & E ) and immunostaining ( red ) showing skin morphology , and T cell ( CD3 + ) and neutrophil ( Ly6b + ) infiltration in WT or IL - 6 - / - mice before ( naïve ) , and 14 days ( H & E , Ly6b + ) , or 21 days ( CD3 + ) post - IR . Infiltrating CD3 + T cells ( yellow arrows ) within degenerating hair follicles ( HF ; dashed lines ) are indicated . Scale bars , 100 μm . Quantification of epidermal thickness at 14 days post - IR ( n = 5 - 8 ) . Kinetics of neutrophil ( Ly6b + ) ( n = 3 - 9 ) and T cell ( CD3 + ) ( n = 3 ) infiltration . Data information : Data represent mean ± SD F , mean ± SD ( G ) . * P < 0 . 05 , * * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 001 by two - tailed Mann - Whitney test F , or two - way ANOVA with Bonferroni ' s posthoc multiple comparisons test ( G ) ; five independent experiments . Quantification by real - time qPCR analysis of Tnfa , Ccl3 , and Ifng mRNAs in whole skin of WT and IL - 6 - / - mice at indicated times post - IR ( 15Gy ) , ( n = 5 - 7 ) . Data information : Data represent mean ± SD H ) * P < 0 . 05 , * * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 001 by , two - tailed Mann - Whitney test H ) , five independent experiments .

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

Figure 2Relative expression and co-localization of IL6 mRNA and IL-6 Signaling Scoring in integrated skin cells by blended UMAPs.Quantification by real-time qPCR analysis of Il6 mRNA in the skin of naïve and irradiated (15Gy) WT mice at indicated times, (n=3-6). Data information: Data represent mean ± SD (B, *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by one-way ANOVA with Dunnet's multiple comparisons test (B), five independent experiments.Quantification of ventral hair loss area 21 days post-IR (15Gy) in WT and IL-6-/- mice, (n=11). Photographic images showing hair loss (area demarked by yellow dashed line) 21 days post-IR (15 Gy) in wild type (WT) and IL-6-/- mice. Data information: Data represent mean ± SD C, *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by , two-tailed Mann-Whitney test (C, ; five independent experiments.Histochemical (H&E) and immunostaining (red) showing skin morphology, and T cell (CD3+) and neutrophil (Ly6b+) infiltration in WT or IL-6-/- mice before (naïve), and 14 days (H&E, Ly6b+), or 21 days (CD3+) post-IR. Infiltrating CD3+ T cells (yellow arrows) within degenerating hair follicles (HF; dashed lines) are indicated. Scale bars, 100 μm. Quantification of epidermal thickness at 14 days post-IR (n = 5-8). Kinetics of neutrophil (Ly6b+) (n = 3-9) and T cell (CD3+) (n = 3) infiltration. Data information: Data represent mean ± SD F, mean ± SD (G). *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by two-tailed Mann-Whitney test F, or two-way ANOVA with Bonferroni's posthoc multiple comparisons test (G); five independent experiments.Quantification by real-time qPCR analysis of Tnfa, Ccl3, and Ifng mRNAs in whole skin of WT and IL-6-/- mice at indicated times post-IR (15Gy), (n = 5-7). Data information: Data represent mean ± SD H) *P<0.05, **P<0.01, **P<0.01, ****P<0.001 by , two-tailed Mann-Whitney test H), five independent experiments.


Figure 3Western blot analysis shows phosphorylated STAT3 ( p - Stat3 ) , Akt ( p - Akt ) ) , Erk1 and Erk2 ( p - Erk1 / 2 ) ) , and β - Actin in the skin of WT , IL - 6 - / - , and IL - 1R - / - mice at indicated times ( days ) post - IR ( 15Gy ) . Histochemical ( H & E ) and immunostaining ( red ) staining of T cells ( CD3 + ) , neutrophils ( Ly6b + ) and phosphorylated Stat3 ( p - Stat3 ) ( red nuclear staining , black arrows ) in skin thin sections prepared at 14 days post - IR from mice treated by daily topical application of acquosum ( control ) , ruxolitinib or tofacitinib following irradiation ( 15 Gy ) . Scale bar , 100 μm . ( CD3 and Ly6b immunostaining images from Tofacitinib treated mice represent seperate analyses performed on serial thin sections . ) Quantification of epidermal thickness , T cells ( CD3 + ) and neutrophils ( Ly6b + ) , and p - STAT3 + cells in ( B ) , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , by two - tailed Student ' s t test ; two independent experiments . Photographic images showing hair loss in mice at 14 days post - IR ( 15 Gy ) following treatment by daily topical application of acquosum ( carrier control ) or JAK inhibitors , ruxolitinib or tofacitinib . Chest and neck hairs of mice in all groups were clipped prior to irradiation to enhance drug absorption . Quantification of the area of ventral hair loss ( in pixels ) in mice in ( D ) , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , by two - tailed Student ' s t test ; two independent experiments .

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

Figure 3Western blot analysis shows phosphorylated STAT3 (p-Stat3), Akt (p-Akt)), Erk1 and Erk2 (p-Erk1/2)), and β-Actin in the skin of WT, IL-6-/-, and IL-1R-/- mice at indicated times (days) post-IR (15Gy).Histochemical (H&E) and immunostaining (red) staining of T cells (CD3+), neutrophils (Ly6b+) and phosphorylated Stat3 (p-Stat3) (red nuclear staining, black arrows) in skin thin sections prepared at 14 days post-IR from mice treated by daily topical application of acquosum (control), ruxolitinib or tofacitinib following irradiation (15 Gy). Scale bar, 100 μm. (CD3 and Ly6b immunostaining images from Tofacitinib treated mice represent seperate analyses performed on serial thin sections.) Quantification of epidermal thickness, T cells (CD3+) and neutrophils (Ly6b+), and p-STAT3+ cells in (B), (n = 4). Data information: Data represent mean ± SD. *P<0.05, **P<0.01, by two-tailed Student's t test; two independent experiments.Photographic images showing hair loss in mice at 14 days post-IR (15 Gy) following treatment by daily topical application of acquosum (carrier control) or JAK inhibitors, ruxolitinib or tofacitinib. Chest and neck hairs of mice in all groups were clipped prior to irradiation to enhance drug absorption. Quantification of the area of ventral hair loss (in pixels) in mice in (D), (n = 4). Data information: Data represent mean ± SD. *P<0.05, **P<0.01, by two-tailed Student's t test; two independent experiments.


Figure 4UMAP plots depicting relative expression and localization of Il1a and Il1b mRNAs in irradiated and naïve skin - derived cell clusters . Photographic images showing hair loss 14 days post - IR ( 15 Gy ) in WT and IL - 1R - / - mice . Quantification of ventral hair loss area 14 days post - IR ( 15Gy ) in WT and IL - 1R - / - mice , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , by two - tailed Mann - Whitney test ( C ) five independent experiments . Histochemical ( H & E ) and immunostaining ( red ) showing morphology and neutrophil ( Ly6b ) infiltration in the skin of mice in ( B ) . Scale bar , 50 μm . Quantification of epidermal thickness , neutrophils ( Ly6b ) and T cells ( CD3 + ) in specimens in ( D ) , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , by Student ' s t test ( E ) ; five independent experiments .

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

Figure 4UMAP plots depicting relative expression and localization of Il1a and Il1b mRNAs in irradiated and naïve skin-derived cell clusters.Photographic images showing hair loss 14 days post-IR (15 Gy) in WT and IL-1R-/- mice. Quantification of ventral hair loss area 14 days post-IR (15Gy) in WT and IL-1R-/- mice, (n = 4). Data information: Data represent mean ± SD. *P<0.05, by two-tailed Mann-Whitney test (C) five independent experiments.Histochemical (H&E) and immunostaining (red) showing morphology and neutrophil (Ly6b) infiltration in the skin of mice in (B). Scale bar, 50 μm. Quantification of epidermal thickness, neutrophils (Ly6b) and T cells (CD3+) in specimens in (D), (n = 4). Data information: Data represent mean ± SD. *P<0.05, by Student's t test (E); five independent experiments.


Figure 5UMAP plots depicting relative expression and localization of Ccr6 and Ccl20 mRNAs in irradiated and naïve skin - derived cell clusters . Quantification by real - time qPCR analysis of Ccr6 and Ccl20 mRNAs in whole skin of naïve and irradiated ( 15Gy ) WT and IL - 6 - / - mice at indicated times post - IR , ( n = 5 - 7 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney test Two independent experiments . Photographic images showing the development of radiodermatitis and alopecia in irradiated ( 15 Gy ) WT and CCR6 - / - mice at indicated times post - IR . Escalating radiodermatitis , which manifests on day 7 - 10 as serous exudates on the chin and neck , is visible in both strains and progresses in WT mice to alopecia by day 14 post - IR but resolves spontaneously in the CCR6 - / - mice . Quantification ( lower right ) of the area of hair loss at 14 days post - IR , ( n = 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney test Two independent experiments . Histochemical staining ( H & E ) and immunostaining ( red ) show morphology and infiltration of neutrophils ( Ly6b + ) and T cells ( CD3 + ) ( red arrows ) to the skin and to the hair follicle ( HF ) ( dashed lines ) in WT or CCR6 - / - mice 14 days post - IR . Scale bars , 50 μm ( black ) and 25 μm ( red ) . Quantification of epidermal thickness , neutrophils ( Ly6b + ) , total T cell ( CD3 + ) infiltration to the skin , and ( F ) CD3 + cells infiltration to the hair follicle in ( D ) , ( n = 5 - 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Student ' s t test ( E - Ly6b , by Mann - Whitney test Two independent experiments . MHC class I immunostaining ( red ) of skin thin sections prepared at 14 days post - IR from mice in ( C ) , and quantification ( right ) of MHC I positive hair follicles , ( n = 4 - 5 ) . Scale bar , 50 μm . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney test Two independent experiments . Quantification by real - time qPCR analysis of Il6 , Il17a , Ccl20 , and Ccl3 mRNAs in the skin of naïve WT and irradiated ( 15Gy ) WT and CCR6 - / - mice at 14 days post - IR , ( n = 3 - 5 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney test Two independent experiments .

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

Figure 5UMAP plots depicting relative expression and localization of Ccr6 and Ccl20 mRNAs in irradiated and naïve skin-derived cell clusters. Quantification by real-time qPCR analysis of Ccr6 and Ccl20 mRNAs in whole skin of naïve and irradiated (15Gy) WT and IL-6-/- mice at indicated times post-IR, (n=5-7). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.Photographic images showing the development of radiodermatitis and alopecia in irradiated (15 Gy) WT and CCR6-/- mice at indicated times post-IR. Escalating radiodermatitis, which manifests on day 7-10 as serous exudates on the chin and neck, is visible in both strains and progresses in WT mice to alopecia by day 14 post-IR but resolves spontaneously in the CCR6-/- mice. Quantification (lower right) of the area of hair loss at 14 days post-IR, (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.Histochemical staining (H&E) and immunostaining (red) show morphology and infiltration of neutrophils (Ly6b+) and T cells (CD3+) (red arrows) to the skin and to the hair follicle (HF) (dashed lines) in WT or CCR6-/- mice 14 days post-IR. Scale bars, 50 μm (black) and 25 μm (red). Quantification of epidermal thickness, neutrophils (Ly6b+), total T cell (CD3+) infiltration to the skin, and (F) CD3+ cells infiltration to the hair follicle in (D), (n = 5-6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t test (E-Ly6b, by Mann-Whitney test Two independent experiments.MHC class I immunostaining (red) of skin thin sections prepared at 14 days post-IR from mice in (C), and quantification (right) of MHC I positive hair follicles, (n = 4-5). Scale bar, 50 μm. Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.Quantification by real-time qPCR analysis of Il6, Il17a, Ccl20, and Ccl3 mRNAs in the skin of naïve WT and irradiated (15Gy) WT and CCR6-/- mice at 14 days post-IR, (n = 3-5). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.


Figure 6Photographic images showing hair loss in control ( PBS ) and cyclosporine ( CsA ) treated WT mice at 14 days post - IR ( 15 Gy ) . Quantification of the area of ventral hair loss in mice in ( A ) , ( n = 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Mann - Whitney test ; two independent experiments . Histochemical ( H & E ) and immunostaining ( red ) show T cell ( CD3 + ) and neutrophil ( Ly6b + ) infiltration in the skin of mice in ( A ) . Scale bar , 100 μm . ( Images of CD3 and Ly6b immunostaining from CsA treated mice represent seperate analyses performed on serial thin sections . ) Quantification of epidermal thickness , and T cell ( CD3 + ) and neutrophil ( Ly6b + ) infiltration ( red staining ) in skin thin sections from ( C ) , ( n = 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Mann - Whitney test ; two independent experiments . Quantification by real - time qPCR analysis of Il6 , Il17a , II22 , Ccl3 , Ccr6 , and Ccl20 mRNAs in whole skin of naïve and irradiated ( 15Gy ) control ( PBS ) and CSA treated WT mice at 14 days post - IR , ( n = 5 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Mann - Whitney test ; two independent experiments .

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

Figure 6Photographic images showing hair loss in control (PBS) and cyclosporine (CsA) treated WT mice at 14 days post-IR (15 Gy). Quantification of the area of ventral hair loss in mice in (A), (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.Histochemical (H&E) and immunostaining (red) show T cell (CD3+) and neutrophil (Ly6b+) infiltration in the skin of mice in (A). Scale bar, 100 μm. (Images of CD3 and Ly6b immunostaining from CsA treated mice represent seperate analyses performed on serial thin sections.) Quantification of epidermal thickness, and T cell (CD3+) and neutrophil (Ly6b+) infiltration (red staining) in skin thin sections from (C), (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.Quantification by real-time qPCR analysis of Il6, Il17a, II22, Ccl3, Ccr6, and Ccl20 mRNAs in whole skin of naïve and irradiated (15Gy) control (PBS) and CSA treated WT mice at 14 days post-IR, (n = 5). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.


Figure 1 I , J . Normal ciliary formation of Rabl2 - / - MEFs . MEFs derived from E12 . 5 mouse embryos were serum - starved for 48 h to induce primary cilia ( I ) . Statistical results ( J ) were from three independent experiments . Ciliogenesis of at least 200 cells was scored in each experiment and condition . Ciliary lengths of 80 cells were measured in each experiment and condition and the results of the same genotypes were pooled together . K , L . Rabl2 deficiency did not repress ciliary localization of IFT - B . Ciliated MEFs were immunostained for Ift81 and Ift57 , two IFT - B subunits ( K ; see Fig 4F for diagram of IFT - B ) . Relative intensity of ciliary Ift81 ( L ) , presented in arbitrary unit , was obtained by normalizing to that of corresponding Ac - tub . Four MEF strains prepared respectively from two embryos per genotype were used for quantification . 50 cilia were quantified from each MEF strain and results of the same genotypes were pooled together .

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

Figure 1 I, J. Normal ciliary formation of Rabl2-/- MEFs. MEFs derived from E12.5 mouse embryos were serum-starved for 48 h to induce primary cilia (I). Statistical results (J) were from three independent experiments. Ciliogenesis of at least 200 cells was scored in each experiment and condition. Ciliary lengths of 80 cells were measured in each experiment and condition and the results of the same genotypes were pooled together. K, L. Rabl2 deficiency did not repress ciliary localization of IFT-B. Ciliated MEFs were immunostained for Ift81 and Ift57, two IFT-B subunits (K; see Fig 4F for diagram of IFT-B). Relative intensity of ciliary Ift81 (L), presented in arbitrary unit, was obtained by normalizing to that of corresponding Ac-tub. Four MEF strains prepared respectively from two embryos per genotype were used for quantification. 50 cilia were quantified from each MEF strain and results of the same genotypes were pooled together.


Figure 2 D , E . Increased Gli3 FL / R ratios in E11 . 5 hind limb buds and E10 . 5 mouse embryos . Relative band intensities of the full - length ( FL ) and the repressor ( R ) forms of Gli3 were quantified ( red ) . FL / R ratios were from four different sets of limb bud preparations ( D ) and three different sets of embryo preparations ( 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 O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 D, E. Increased Gli3 FL/R ratios in E11.5 hind limb buds and E10.5 mouse embryos. Relative band intensities of the full-length (FL) and the repressor (R) forms of Gli3 were quantified (red). FL/R ratios were from four different sets of limb bud preparations (D) and three different sets of embryo preparations (E).


Figure 3 Rabl2Q80L inhibited Hh signaling . MEFs from Rabl2 - / - E12 . 5 embryos infected with lentivirus to express GFP , GFP - Rabl2 or GFP - Rabl2Q80L were serum - starved for 24 h and treated with DMSO ( mock ) or 100 nM SAG for an additional 24 h . Uninfected MEFs from the wildtype littermate embryos served as the positive control . The samples were immunostained to examine ciliary Gli2 The white arrowheads ( G ) point to ciliary tips . Quantification results were from three independent experiments , each using MEFs from different Rabl2 - deficient embryos . Gli2 intensities , presented in arbitrary unit , were measured from at least 22 cilia in each experiment and condition and pooled together . H . Rabl2Q80L inhibited Hh signaling . MEFs from Rabl2 - / - E12 . 5 embryos infected with lentivirus to express GFP , GFP - Rabl2 or GFP - Rabl2Q80L were serum - starved for 24 h and treated with DMSO ( mock ) or 100 nM SAG for an additional 24 h . Uninfected MEFs from the wildtype littermate embryos served as the positive control . The samples were immunoblotted to examine levels of Gli1 and Gli3 In the samples for immunoblotting ( H ) , GFP - positive cells occupied 82 . 3 % ( GFP ) , 87 . 3 % ( GFP - Rabl2 ) , and 81 . 2 % ( GFP - Rabl2Q80L ) , respectively , when 260 cells in each sample were scored . Relative band intensities are shown in red .

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

Figure 3 Rabl2Q80L inhibited Hh signaling. MEFs from Rabl2-/- E12.5 embryos infected with lentivirus to express GFP, GFP-Rabl2 or GFP-Rabl2Q80L were serum-starved for 24 h and treated with DMSO (mock) or 100 nM SAG for an additional 24 h. Uninfected MEFs from the wildtype littermate embryos served as the positive control. The samples were immunostained to examine ciliary Gli2 The white arrowheads (G) point to ciliary tips. Quantification results were from three independent experiments, each using MEFs from different Rabl2-deficient embryos. Gli2 intensities, presented in arbitrary unit, were measured from at least 22 cilia in each experiment and condition and pooled together.  H. Rabl2Q80L inhibited Hh signaling. MEFs from Rabl2-/- E12.5 embryos infected with lentivirus to express GFP, GFP-Rabl2 or GFP-Rabl2Q80L were serum-starved for 24 h and treated with DMSO (mock) or 100 nM SAG for an additional 24 h. Uninfected MEFs from the wildtype littermate embryos served as the positive control. The samples were immunoblotted to examine levels of Gli1 and Gli3 In the samples for immunoblotting (H), GFP-positive cells occupied 82.3% (GFP), 87.3% (GFP-Rabl2), and 81.2% (GFP-Rabl2Q80L), respectively, when 260 cells in each sample were scored. Relative band intensities are shown in red.


Figure 4 I . Association of endogenous Rabl2 - GTP with IFT - B . Mouse testis lysates with or without 1 mM GTPγS were subjected to sucrose gradient ultracentrifugation . Fractions were collected from top ( low MW ) to bottom ( high MW ) . Note the obvious enrichment of Rabl2 in fraction # 16 in the presence of GTPγS .

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Figure 4 I. Association of endogenous Rabl2-GTP with IFT-B. Mouse testis lysates with or without 1 mM GTPγS were subjected to sucrose gradient ultracentrifugation. Fractions were collected from top (low MW) to bottom (high MW). Note the obvious enrichment of Rabl2 in fraction #16 in the presence of GTPγS.


Figure 5 D , E . Ciliary accumulation of BBS proteins in Rabl2Q80L - expressing RPE1 cells . The white arrows ( D ) point to positions of cilia . Quantification results ( E ) were from three independent experiments . At least 100 GFP - positive cells were scored in each experiment and condition . H , I . BBSomes and IFT - B were able to reach the ciliary base through retrograde IFT in the presence of Rabl2Q80L . Ciliary 3NG - Bbs5 ( H ) or Ift27 - GFP ( I ) in IMCD3 cells co - expressing RFP - Rabl2 or RFP - Rabl2Q80L was live imaged at 4 fps . Snapshots for the ciliary RFP proteins , the first frames from Movies EV2 - EV5 , corresponding kymographs , and quantification results from the indicated numbers of cilia are presented . For processivity analysis ( I ) , only traceable puncta were scored .

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Figure 5 D, E. Ciliary accumulation of BBS proteins in Rabl2Q80L-expressing RPE1 cells. The white arrows (D) point to positions of cilia. Quantification results (E) were from three independent experiments. At least 100 GFP-positive cells were scored in each experiment and condition.  H, I. BBSomes and IFT-B were able to reach the ciliary base through retrograde IFT in the presence of Rabl2Q80L. Ciliary 3NG-Bbs5 (H) or Ift27-GFP (I) in IMCD3 cells co-expressing RFP-Rabl2 or RFP-Rabl2Q80L was live imaged at 4 fps. Snapshots for the ciliary RFP proteins, the first frames from Movies EV2-EV5, corresponding kymographs, and quantification results from the indicated numbers of cilia are presented. For processivity analysis (I), only traceable puncta were scored.


Figure 6 E , F . Rabl2D73GQ80L did not repress ciliary translocation of SMO and GPR161 . RPE1 cells expressing GFP - tagged Rabl2Q80L or Rabl2D73GQ80L were serum - starved for 24 h and treated with DMSO ( mock ) or SAG for an additional 24 h prior to immunostaining ( E ) . The white arrows ( E ) point to positions of cilia . In quantification results ( F ) , at least 100 cells were scored in each experiment and condition . G . Ciliary Rabl2D73GQ80L did not repress the BBSome export . FLIP assays were performed in RPE1 cells co - expressing 3NG - Bbs5 with RFP - tagged Rabl2Q80L or Rabl2D73GQ80L as depicted in Fig 5F . Photobleaching was executed at t = 0 . Quantification results on half - life ( t1 / 2 ) were from 10 cilia for each condition .

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Figure 6 E, F. Rabl2D73GQ80L did not repress ciliary translocation of SMO and GPR161. RPE1 cells expressing GFP-tagged Rabl2Q80L or Rabl2D73GQ80L were serum-starved for 24 h and treated with DMSO (mock) or SAG for an additional 24 h prior to immunostaining (E). The white arrows (E) point to positions of cilia. In quantification results (F), at least 100 cells were scored in each experiment and condition.  G. Ciliary Rabl2D73GQ80L did not repress the BBSome export. FLIP assays were performed in RPE1 cells co-expressing 3NG-Bbs5 with RFP-tagged Rabl2Q80L or Rabl2D73GQ80L as depicted in Fig 5F. Photobleaching was executed at t = 0. Quantification results on half-life (t1/2) were from 10 cilia for each condition.


Figure 7 F , G . Endogenous Rabl2Q80L entered ciliary shaft to repress the export of the BBSome ( Bbs5 ) and its cargo GPCR ( Smo ) . mEPCs cultured from Rabl2KI / KI and wildtype neonatal mice were fixed at day 7 post serum starvation . Anti - Rabl2 antibody was used to visualize Rabl2 and Rabl2Q80L . Ac - tub or Arl13b served as ciliary marker . Representative micrographs of multicilia ( F ) and primary cilia ( G ) are shown . The white arrows point to positions of cilia .

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Figure 7 F, G. Endogenous Rabl2Q80L entered ciliary shaft to repress the export of the BBSome (Bbs5) and its cargo GPCR (Smo). mEPCs cultured from Rabl2KI/KI and wildtype neonatal mice were fixed at day 7 post serum starvation. Anti-Rabl2 antibody was used to visualize Rabl2 and Rabl2Q80L. Ac-tub or Arl13b served as ciliary marker. Representative micrographs of multicilia (F) and primary cilia (G) are shown. The white arrows point to positions of cilia.


Figure 1Detection of phosphorylated 17K in BYDV - infected cells using Phos - tag SDS - PAGE - immunoblotting assays with an anti - 17K antibody . dpi , days after BYDV inoculation ; P , phosphorylated 17K bands ; λ - PP , λ - phosphatase . Active HvSnRK1 , but not its total amount , was increased in BYDV - infected barley plants as compared to the mock controls . P - SnRK1 or SnRK1 , enzymatically active SnRK1 ( with T172 phosphorylation ) or unphosphorylated SnRK1 protein . Analysis of 17K phosphorylation by HvGRIK1 - HvSnRK1 kinases in vitro . The kinase reactions were set up by mixing the desired GST fusions of 17K , 17K3A , 17K5A , HvGRIK1 , and HvSnRK1 kinase domain ( KD ) , with the reaction products analyzed using Phos - tag SDS - PAGE - immunoblotting assays with an anti - 17K antibody . P , phosphorylated 17K bands ; arrowhead , unphosphorylated 17K .

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Figure 1Detection of phosphorylated 17K in BYDV-infected cells using Phos-tag SDS-PAGE-immunoblotting assays with an anti-17K antibody. dpi, days after BYDV inoculation; P, phosphorylated 17K bands; λ-PP, λ-phosphatase.Active HvSnRK1, but not its total amount, was increased in BYDV-infected barley plants as compared to the mock controls. P-SnRK1 or SnRK1, enzymatically active SnRK1 (with T172 phosphorylation) or unphosphorylated SnRK1 protein.Analysis of 17K phosphorylation by HvGRIK1-HvSnRK1 kinases in vitro. The kinase reactions were set up by mixing the desired GST fusions of 17K, 17K3A, 17K5A, HvGRIK1, and HvSnRK1 kinase domain (KD), with the reaction products analyzed using Phos-tag SDS-PAGE-immunoblotting assays with an anti-17K antibody. P, phosphorylated 17K bands; arrowhead, unphosphorylated 17K.


Figure 2Confirmation of the expression of SnRK1K139R - YFP or SnRK1 - YFP fusion protein each in two transgenic lines , SnRK1K139R - YFP - L13 and - L22 or SnRK1 - YFP - L4 and - L17 , by immunoblotting . The protein samples analyzed here or in ( B ) and ( C ) were all prepared from BYDV - infected barley plants at 21 dpi . Phosphorylation of BYDV 17K was increased in SnRK1 - YFP - L4 plants overexpressing SnRK1 - YFP but reduced in SnRK1K139R - YFP - L13 ectopically expressing SnRK1K139R - YFP compared with WT control . P , phosphorylated 17K bands . Quantitative comparisons of plant height ( D ) , maximum root length ( E ) among the set of BYDV - infected barley materials shown in ( C ) ( using the samples collected at 21 dpi ) . protein ( H ) levels of viral CP gene in the set of BYDV - infected barley materials shown in ( C ) ( using the samples collected at 21 dpi ) .

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Figure 2Confirmation of the expression of SnRK1K139R-YFP or SnRK1-YFP fusion protein each in two transgenic lines, SnRK1K139R-YFP-L13 and -L22 or SnRK1-YFP-L4 and -L17, by immunoblotting. The protein samples analyzed here or in (B) and (C) were all prepared from BYDV-infected barley plants at 21 dpi.Phosphorylation of BYDV 17K was increased in SnRK1-YFP-L4 plants overexpressing SnRK1-YFP but reduced in SnRK1K139R-YFP-L13 ectopically expressing SnRK1K139R-YFP compared with WT control. P, phosphorylated 17K bands.Quantitative comparisons of plant height (D), maximum root length (E) among the set of BYDV-infected barley materials shown in (C) (using the samples collected at 21 dpi).protein (H) levels of viral CP gene in the set of BYDV-infected barley materials shown in (C) (using the samples collected at 21 dpi).


Figure 4Levels of GFP siRNA detected by sRNA blotting for the panel of tobacco materials shown in ( A ) , with arrowhead indicating the main band of GFP siRNA . Protein levels of PEBV CP and corrected expression of 17K , 17K5A or 17K5D in the panel of tobacco plants shown in ( D ) , revealed using immunoblotting with anti - PEBV CP or anti - 17K antibody .

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Figure 4Levels of GFP siRNA detected by sRNA blotting for the panel of tobacco materials shown in (A), with arrowhead indicating the main band of GFP siRNA.Protein levels of PEBV CP and corrected expression of 17K, 17K5A or 17K5D in the panel of tobacco plants shown in (D), revealed using immunoblotting with anti-PEBV CP or anti-17K antibody.


Figure 517K5D , but not BYDV 17K , interacted with HvSDN1 in LC ( B ) assays . Expression of 17K and 17K5D in the LC assays were verified by immunoblotting using anti - 17K antibody . C 17K5D , but not 17K or 17K5A , showed binding to BYDV vsiRNA . The GST fusions of 17K , 17K5D , and 17K5A , as well as free GST , were used in the RNA EMSA assays with biotin - labeled vsiRNA899 or vsiRNA899 duplex as probes .

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Figure 517K5D, but not BYDV 17K, interacted with HvSDN1 in LC (B) assays. Expression of 17K and 17K5D in the LC assays were verified by immunoblotting using anti-17K antibody.C 17K5D, but not 17K or 17K5A, showed binding to BYDV vsiRNA. The GST fusions of 17K, 17K5D, and 17K5A, as well as free GST, were used in the RNA EMSA assays with biotin-labeled vsiRNA899 or vsiRNA899 duplex as probes.


Figure 6Cleavage of BYDV vsiRNA899 by HvSDN1 ( His - HvSDN1 ) , with AtSDN1 ( His - AtSDN1 ) serving as a positive control . Asterisk denotes cleavage products . The GST fusions of 17K and 17K5D did not show vsiRNA cleavage activity . Inhibition of HvSDN1 - catalyzed vsiRNA cleavage by GST - 17K5D but not GST - 17K . Free GST protein served as a negative control . Asterisk denotes cleavage products . The three mutants of 17K5D ( 17K5Dm4 , 17K5Dm2a , and 17K5Dm2b ) retained the ability to interact with HvSDN1 but their GST fusions failed to bind vsiRNA899 ( D ) . Neither GST nor GST - 17K showed vsiRNA899 binding . Quantitative analysis of HvSDN1 cleavage activities , with the fluorescence recorded using Typhoon FLA9500 ( F and G ) Compared with free GST or GST - 17K , the GST fusions of 17K5D or three derivative mutants ( 17K5Dm4 , 17K5Dm2a , and 17K5Dm2b ) inhibited HvSDN1 - catalzyed cleavage of fluorogenic vsiRNA899 , which was revealed by a fluorospot assay ( F ) , relative comparison of fluorescence values ( G )

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Figure 6Cleavage of BYDV vsiRNA899 by HvSDN1 (His-HvSDN1), with AtSDN1 (His-AtSDN1) serving as a positive control. Asterisk denotes cleavage products. The GST fusions of 17K and 17K5D did not show vsiRNA cleavage activity.Inhibition of HvSDN1-catalyzed vsiRNA cleavage by GST-17K5D but not GST-17K. Free GST protein served as a negative control. Asterisk denotes cleavage products.The three mutants of 17K5D (17K5Dm4, 17K5Dm2a, and 17K5Dm2b) retained the ability to interact with HvSDN1 but their GST fusions failed to bind vsiRNA899 (D). Neither GST nor GST-17K showed vsiRNA899 binding.Quantitative analysis of HvSDN1 cleavage activities, with the fluorescence recorded using Typhoon FLA9500 (F and G) Compared with free GST or GST-17K, the GST fusions of 17K5D or three derivative mutants (17K5Dm4, 17K5Dm2a, and 17K5Dm2b) inhibited HvSDN1-catalzyed cleavage of fluorogenic vsiRNA899, which was revealed by a fluorospot assay (F), relative comparison of fluorescence values (G)


Figure 7Co - IP assays revealing the presence of vsiRNA - containing HvAGO1 - HvSDN1 - P17K complex in BYDV - infected WT , SnRK1K139R - YFP - L13 , and SnRK1 - YFP - L4 plants , with mock - inoculated SnRK1 - YFP - L4 individuals as controls . The immunoprecipitates , prepared using anti - HvAGO1 antibody , were analyzed for the presence of HvSDN1 ( by immunoblotting with anti - HvSDN1 antibody ) , P17K ( asterisk ) and 17K ( arrowhead ) ( using Phos - tag SDS - PAGE coupled immunoblotting ) , and BYDV vsiRNA ( by sRNA blotting ) . Compared with WT control , the HvAGO1 immunoprecipitates derived from SnRK1 - YFP - L4 contained higher levels of P17K and vsiRNA but less amount of HvSDN1 , whereas the reverse was observed for SnRK1K139R - YFP - L13 . Co - IP assays demonstrating that the interaction between HvAGO1 and HvSDN1 was weakened by P17K . The immunoprecipitates were prepared using anti - HvSDN1 antibody from the panel of plants shown in ( B ) . Relative to WT control , the HvSDN1 immunoprecipitates derived from SnRK1 - YFP - L4 contained much less HvAGO1 , whereas the opposite was observed for SnRK1K139R - YFP - L13 . 17K5D compromised the interaction between HvSDN1 and the PAZ domain of HvAGO1 . The LUC signal resulted from the interaction between HvSDN1 - HvAGO1 PAZ domain was significantly decreased in the presence of 17K5D but not that of 17K .

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Figure 7Co-IP assays revealing the presence of vsiRNA-containing HvAGO1-HvSDN1-P17K complex in BYDV-infected WT, SnRK1K139R-YFP-L13, and SnRK1-YFP-L4 plants, with mock-inoculated SnRK1-YFP-L4 individuals as controls. The immunoprecipitates, prepared using anti-HvAGO1 antibody, were analyzed for the presence of HvSDN1 (by immunoblotting with anti-HvSDN1 antibody), P17K (asterisk) and 17K (arrowhead) (using Phos-tag SDS-PAGE coupled immunoblotting), and BYDV vsiRNA (by sRNA blotting). Compared with WT control, the HvAGO1 immunoprecipitates derived from SnRK1-YFP-L4 contained higher levels of P17K and vsiRNA but less amount of HvSDN1, whereas the reverse was observed for SnRK1K139R-YFP-L13.Co-IP assays demonstrating that the interaction between HvAGO1 and HvSDN1 was weakened by P17K. The immunoprecipitates were prepared using anti-HvSDN1 antibody from the panel of plants shown in (B). Relative to WT control, the HvSDN1 immunoprecipitates derived from SnRK1-YFP-L4 contained much less HvAGO1, whereas the opposite was observed for SnRK1K139R-YFP-L13.17K5D compromised the interaction between HvSDN1 and the PAZ domain of HvAGO1. The LUC signal resulted from the interaction between HvSDN1-HvAGO1 PAZ domain was significantly decreased in the presence of 17K5D but not that of 17K.


Figure 8Verification of 17K5D expression in the transgenic lines 17K5D - L3 and - L7 by immunoblotting with anti - 17K antibody . B - D Comparison of BYDV disease symptoms including plant growth ( B ) , plant height ( C ) , and maximum root length ( D ) between two transgenic lines and WT control at 21 dpi . protein ( F ) levels of viral CP gene in the BYDV - infected samples shown in ( B ) . BYDV vsiRNA abundance in the set of samples shown in ( B ) , as detected by sRNA blotting at 14 dpi . Relative to TaSDN1 - DD line , TaSDN1 protein was decreased in TaSDN1 - Dd and TaSDN1 - dd mutants , with a stronger reduction found for TaSDN1 - dd . The reactive TaSDN1 protein detected in TaSDN1 - dd was caused by the un - mutated TaSDN1 - A and - B homoeologs . protein ( M ) levels of BYDV CP gene in TaSDN1 - DD , TaSDN1 - Dd and TaSDN1 - dd plants at 21 dpi . BYDV vsiRNA abundance in TaSDN1 - DD , TaSDN1 - Dd , and TaSDN1 - dd plants detected by sRNA blotting at 21 dpi of BYDV .

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Figure 8Verification of 17K5D expression in the transgenic lines 17K5D-L3 and -L7 by immunoblotting with anti-17K antibody.B-D Comparison of BYDV disease symptoms including plant growth (B), plant height (C), and maximum root length (D) between two transgenic lines and WT control at 21 dpi.protein (F) levels of viral CP gene in the BYDV-infected samples shown in (B). BYDV vsiRNA abundance in the set of samples shown in (B), as detected by sRNA blotting at 14 dpi.Relative to TaSDN1-DD line, TaSDN1 protein was decreased in TaSDN1-Dd and TaSDN1-dd mutants, with a stronger reduction found for TaSDN1-dd. The reactive TaSDN1 protein detected in TaSDN1-dd was caused by the un-mutated TaSDN1-A and -B homoeologs.protein (M) levels of BYDV CP gene in TaSDN1-DD, TaSDN1-Dd and TaSDN1-dd plants at 21 dpi. BYDV vsiRNA abundance in TaSDN1-DD, TaSDN1-Dd, and TaSDN1-dd plants detected by sRNA blotting at 21 dpi of BYDV.


Figure 1 A Ex vivo flow cytometry on LN cells of mice 7 days after two s . c . immunizations ( 14 days apart ) with PBS + DMSO ( Veh ) , KLH + DMSO ( KLH ) , KLH + IL - 2 , KLH + RA , KLH + RA + IL - 2 . Results are representative FACS plots of LAG3 + CD4 + or CD25 + Foxp3 + cells after gating on live single CD3 + CD4 + CD49b + Foxp3 - and live single CD3 + CD4 + lymphocytes respectively . C Expression of Tr1 markers PD - 1 , CD226 , ICOS , CD25 and CTLA - 4 on CD49b + LAG - 3 + Foxp3 - CD4 + T cells or on CD49b - CD4 + T cells . D IL - 10 , IL - 17 and IFN - γ production by ELISA on supernatants of spleen cells from mice immunized with PBS + DMSO ( Veh ) , KLH + DMSO ( KLH ) , KLH + RA , KLH + IL - 2 , KLH + RA + IL - 2 or KLH + LP - GMP ( LP1569 + c - di - GMP ) and cultured for 5 days with increasing concentrations of KLH . Bars are mean + SEM ( n = 4 or 5 ) . * * p < 0 . 01 , * * * p < 0 . 001 , KLH + IL - 2 + RA vs KLH , # # p < 0 . 01 , # # # p < 0 . 001 KLH + IL - 2 + RA vs KLH + RA , † † † p < 0 . 001 KLH + IL - 2 + RA vs KLH + IL - 2 by two - way ANOVA and Tukey post hoc test .

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

Figure 1 A Ex vivo flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + IL-2, KLH + RA, KLH + RA + IL-2. Results are representative FACS plots of LAG3+CD4+ or CD25+Foxp3+ cells after gating on live single CD3+CD4+CD49b+Foxp3- and live single CD3+CD4+ lymphocytes respectively.C Expression of Tr1 markers PD-1, CD226, ICOS, CD25 and CTLA-4 on CD49b+ LAG-3+ Foxp3-CD4+ T cells or on CD49b-CD4+ T cells.D IL-10, IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice immunized with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + RA, KLH + IL-2, KLH + RA + IL-2 or KLH + LP-GMP (LP1569 + c-di-GMP) and cultured for 5 days with increasing concentrations of KLH. Bars are mean + SEM (n=4 or 5). **p<0.01, ***p<0.001, KLH + IL-2 + RA vs KLH, ##p<0.01, ###p <0.001 KLH + IL-2 + RA vs KLH + RA, †††p<0.001 KLH + IL-2 + RA vs KLH + IL-2 by two-way ANOVA and Tukey post hoc test.


Figure 2 A Flow cytometry on LN cells of mice 7 days after two s . c . immunizations ( 14 days apart ) with PBS + DMSO ( Veh ) , MOG + DMSO ( MOG ) , MOG + RA + IL - 2 . Cells were gated on live single CD3 + lymphocytes . FMO : Fluorescence Minus One . B Mean percentages of IL - 10 + , LAG - 3 + , PD1 + or Foxp3 + CD25 + CD4 T cells as described in A . Cells were gated on live single CD3 + lymphocytes ( top left panel ) or on live single CD3 + CD4 + lymphocytes . Bars are mean + SEM ( n = 5 ) . * p < 0 . 05 , * * * p < 0 . 01 by one - way ANOVA and Tukey post hoc test . C EAE clinical scores in mice immunized s . c . with MOG + RA , MOG + IL - 2 , MOG + RA + IL - 2 , or PBS + DMSO ( Veh ) , 7 and 21 days before induction of EAE . Results are mean + SEM ( n = 12 , except for MOG + IL - 2 , where n = 6 ; combined from two experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , MOG + IL - 2 + RA vs Veh , # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 , MOG + IL - 2 + RA vs MOG + RA , † p < 0 . 05 , † † p < 0 . 01 , † † † p < 0 . 001 , MOG + IL - 2 + RA vs MOG + IL - 2 by two - way ANOVA and Tukey post hoc test . D Representative FACS plots of MOG dextramer + CD4 T cells in LNs of naïve mice or 3 days after induction of EAE . Cells were gated as single live F4 / 80 - CD8 - B220 - CD4 + CD44 + . E Mean percentages of MOG dextramer + CD4 T cells in LNs 3 days after induction of EAE . Cells were gated as single live F4 / 80 - CD8 - B220 - CD4 + CD44 + . Bars are mean + SEM ( n = 5 or 6 ) . * p < 0 . 05 by unpaired two - tailed t test . F Mean fluorescence intensity ( MFI ) of CD49b , ICOS and PD - 1 by MOG - dextramer + CD4 T cells in LNs 3 days after induction of EAE . Cells were gated as single live F4 / 80 - CD8 - B220 - CD4 + CD44 + dextramer + . Bars are mean + SEM ( n = 5 or 6 ) . * p < 0 . 05 , * * p < 0 . 01 by unpaired two - tailed t test . G MOG - specific IL - 17 and IFN - γ production by spleen cells from day 7 of EAE , quantified in supernatants by ELISA after 5 d culture with increasing concentrations of MOG . Bars are mean + SEM ( n = 3 or 4 ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - ANOVA and Tukey post hoc test .

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

Figure 2 A Flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), MOG + DMSO (MOG), MOG + RA + IL-2. Cells were gated on live single CD3+ lymphocytes. FMO: Fluorescence Minus One. B Mean percentages of IL-10+, LAG-3+, PD1+ or Foxp3+ CD25+ CD4 T cells as described in A. Cells were gated on live single CD3+ lymphocytes (top left panel) or on live single CD3+ CD4+ lymphocytes. Bars are mean + SEM (n=5). *p <0.05, ***p <0.01 by one-way ANOVA and Tukey post hoc test.  C EAE clinical scores in mice immunized s.c. with MOG + RA, MOG + IL-2, MOG + RA + IL-2, or PBS + DMSO (Veh), 7 and 21 days before induction of EAE. Results are mean + SEM (n=12, except for MOG + IL-2, where n=6; combined from two experiments). *p<0.05, **p<0.01, ***p <0.001, MOG + IL-2 + RA vs Veh, #p<0.05, ##p<0.01, ###p<0.001, MOG + IL-2 + RA vs MOG + RA, †p<0.05, ††p<0.01, †††p <0.001, MOG + IL-2 + RA vs MOG + IL-2 by two-way ANOVA and Tukey post hoc test.  D Representative FACS plots of MOG dextramer+ CD4 T cells in LNs of naïve mice or 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+.  E Mean percentages of MOG dextramer+ CD4 T cells in LNs 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+. Bars are mean + SEM (n=5 or 6). *p <0.05 by unpaired two-tailed t test. F Mean fluorescence intensity (MFI) of CD49b, ICOS and PD-1 by MOG-dextramer+ CD4 T cells in LNs 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+ dextramer+. Bars are mean + SEM (n=5 or 6). *p <0.05, **p <0.01 by unpaired two-tailed t test. G MOG-specific IL-17 and IFN-γ production by spleen cells from day 7 of EAE, quantified in supernatants by ELISA after 5 d culture with increasing concentrations of MOG. Bars are mean + SEM (n=3 or 4). *p<0.05, **p<0.01 and ***p<0.001 by two-ANOVA and Tukey post hoc test.


Figure 3 A Representative FACS plots of CD11a + CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG , RA and IL - 2 or vehicle ( Veh ) only . Cells were gated as single live CD45 + CD3 + CD4 + . B Mean absolute numbers of CD11a + CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG , RA and IL - 2 . Bars are mean + SEM ( n = 4 or 5 ) . * p < 0 . 05 by unpaired two - tailed t test . C Representative FACS plots of CD45 + CD4 T cells in the spinal cords of mice on day 12 of EAE . Cells were gated as single live CD45 + . D Mean absolute numbers of infiltrating CD45 + cells in the spinal cords of mice on day 12 of EAE . Bars are mean + SEM ( n = 5 or 6 ) . * p < 0 . 05 by unpaired two - tailed t test . E Representative FACS plots of infiltrating IL - 17 + and IFN - γ + CD4 T cells in the spinal cords on day 12 of EAE . Cells were gated as single live CD45 + CD3 + CD4 + . F Mean absolute numbers of infiltrating IL - 17 + and IFN - γ + CD4 T cells in the spinal cords on day 12 of EAE . Bars are mean + SEM ( n = 5 or 6 ) . * p < 0 . 05 by unpaired two - tailed t test . G Representative histogram of LAG - 3 expression on CD4 T cells in the spinal cords on day 12 of EAE . Cells were gated as single live CD45 + CD3 + CD4 + . H Inverse correlation between LAG - 3 and IL - 17 and IFN - γ expression on CD4 T cells in the spinal cords on day 12 of EAE . * p < 0 . 05 by unpaired two - tailed t test .

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

Figure 3 A Representative FACS plots of CD11a+ CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG, RA and IL-2 or vehicle (Veh) only. Cells were gated as single live CD45+ CD3+ CD4+. B Mean absolute numbers of CD11a+ CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG, RA and IL-2. Bars are mean + SEM (n=4 or 5). *p<0.05 by unpaired two-tailed t test.  C Representative FACS plots of CD45+ CD4 T cells in the spinal cords of mice on day 12 of EAE. Cells were gated as single live CD45+. D Mean absolute numbers of infiltrating CD45+ cells in the spinal cords of mice on day 12 of EAE. Bars are mean + SEM (n=5 or 6). *p<0.05 by unpaired two-tailed t test.  E Representative FACS plots of infiltrating IL-17+ and IFN-γ+ CD4 T cells in the spinal cords on day 12 of EAE. Cells were gated as single live CD45+ CD3+ CD4+. F Mean absolute numbers of infiltrating IL-17+ and IFN-γ+ CD4 T cells in the spinal cords on day 12 of EAE. Bars are mean + SEM (n=5 or 6). *p<0.05 by unpaired two-tailed t test.  G Representative histogram of LAG-3 expression on CD4 T cells in the spinal cords on day 12 of EAE. Cells were gated as single live CD45+ CD3+ CD4+.  H Inverse correlation between LAG-3 and IL-17 and IFN-γ expression on CD4 T cells in the spinal cords on day 12 of EAE. *p<0.05 by unpaired two-tailed t test.