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

(K) 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.


( 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 .

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

(A-C) 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 .

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

(D-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 .

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

(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 .

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

(K-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(N) Red and green arrowheads in (N) point to a SOX2+ (red) progenitor and a TUJ1+ (blue) neuron, respectively. Scale bars: 50µm.


Relative expression and co - localization of IL6 mRNA and IL - 6 Signaling Scoring in integrated skin cells by blended UMAPs .

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

Relative 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 .

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

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 .

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

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 .

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

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

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.


Western 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 ) .

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

Western 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 .

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

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

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.


UMAP plots depicting relative expression and localization of Il1a and Il1b mRNAs in irradiated and naïve skin - derived cell clusters .

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

UMAP 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 .

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

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

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.


UMAP 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 .

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

UMAP 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 .

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

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 .

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

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 .

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

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

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.


Photographic 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 .

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Photographic 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 .

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

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.


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

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.


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 .

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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.


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 ) .

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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).


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 .

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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 .

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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.


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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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.


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 .

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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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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.


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 .

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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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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.


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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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.


Detection 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 .

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Detection 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 .

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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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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.


Confirmation 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 .

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Confirmation 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 .

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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 ) .

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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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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).


Levels 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 .

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Levels 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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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.


17K5D , 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 .

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17K5D, 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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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.


Cleavage 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 .

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Cleavage 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 .

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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 .

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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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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)


Co - 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 .

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

Co-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 .

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

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 .

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.


Verification of 17K5D expression in the transgenic lines 17K5D - L3 and - L7 by immunoblotting with anti - 17K antibody .

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

Verification 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 .

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 ) .

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

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 .

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

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 .

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

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 .

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

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 .

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

BYDV vsiRNA abundance in TaSDN1-DD, TaSDN1-Dd, and TaSDN1-dd plants detected by sRNA blotting at 21 dpi of BYDV.


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 .

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

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.


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 +

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

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+


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 .

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

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

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.


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 .

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

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 .

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

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 + .

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

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 .

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

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 .

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

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

G 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.


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 .

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

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.


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 + .

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

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+.


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 .

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

C 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.


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 + .

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

C 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+.


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 .

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

E 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.


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 + .

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

E 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+.


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 + .

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

G 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

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.


A IL - 17 and IFN - γ production by ELISA on supernatants of spleen cells from mice with EAE ( EAE ) co - cultured for 3 days with MOG - specific Tr1 cells ( from mice immunized twice with MOG , RA and IL - 2 and amplified in vitro with MOG , RA and IL - 2 for 8 days ) or control T cells from LNs of mice injected with PBS + DMSO ( Veh ) . Bars are mean + SD of one experiment . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 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 B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice with EAE (EAE) co-cultured for 3 days with MOG-specific Tr1 cells (from mice immunized twice with MOG, RA and IL-2 and amplified in vitro with MOG, RA and IL-2 for 8 days) or control T cells from LNs of mice injected with PBS + DMSO (Veh). Bars are mean + SD of one experiment. *p<0.05, **p<0.01 and ***p<0.001 by unpaired two-tailed t test.


B EAE clinical scores of mice transferred 1 day before induction of EAE with MOG - specific Tr1 cells amplified in vitro with MOG , RA and IL - 2 . Results are mean + SEM ( n = 4 or 6 ) . * p < 0 . 05 and * * p < 0 . 01 by two - way ANOVA with Bonferroni post hoc test .

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

B EAE clinical scores of mice transferred 1 day before induction of EAE with MOG-specific Tr1 cells amplified in vitro with MOG, RA and IL-2. Results are mean + SEM (n=4 or 6). *p<0.05 and **p<0.01 by two-way ANOVA with Bonferroni post hoc test.


C Representative FACS plots of IL - 17 + CD4 T cells in the spinal cords on day 20 of EAE . Cells were gated as live single CD45 . 2 + CD3 + CD4 + cells . D Mean absolute numbers of IL - 17 + CD4 T cells in the spinal cords on day 20 of EAE . Results are mean + SEM ( n = 6 ) . * 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

C Representative FACS plots of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Cells were gated as live single CD45.2+CD3+CD4+ cells. D Mean absolute numbers of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Results are mean + SEM (n=6). *p<0.05 by unpaired two-tailed t test.


C Representative FACS plots of IL - 17 + CD4 T cells in the spinal cords on day 20 of EAE . Cells were gated as live single CD45 . 2 + CD3 + CD4 + cells .

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

C Representative FACS plots of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Cells were gated as live single CD45.2+CD3+CD4+ cells.


E Representative FACS plots of CTLA - 4 + CD4 T cells in the spleens of mice with EAE . Cells were gated as live single CD45 . 1 + CD3 + CD4 + . F Mean percentages of CTLA - 4 + CD4 T cells in the spleens of mice with EAE . Results are mean + SEM ( n = 6 ) . * * p < 0 . 01 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

E Representative FACS plots of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Cells were gated as live single CD45.1+CD3+CD4+. F Mean percentages of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Results are mean + SEM (n=6). **p<0.01 by unpaired two-tailed t test.


E Representative FACS plots of CTLA - 4 + CD4 T cells in the spleens of mice with EAE . Cells were gated as live single CD45 . 1 + CD3 + CD4 + .

E Representative FACS plots of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Cells were gated as live single CD45.1+CD3+CD4+.


G EAE clinical scores of WT and IL - 10 - / - mice immunized s . c . with MOG + RA + IL - 2 , or PBS + DMSO ( Veh ) , 7 and 21 days before induction of EAE . Results are mean + SEM ( n = 6 or 7 ) . * * * p < 0 . 001 , WT MOG + IL - 2 + RA vs WT Veh , # # p < 0 . 01 , # # # p < 0 . 001 , IL - 10 - / - MOG + IL - 2 + RA vs IL - 10 - / - Veh 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

G EAE clinical scores of WT and IL-10-/- mice immunized s.c. with MOG + RA + IL-2, or PBS + DMSO (Veh), 7 and 21 days before induction of EAE. Results are mean + SEM (n=6 or 7). ***p <0.001, WT MOG + IL-2 + RA vs WT Veh, ##p<0.01, ###p<0.001, IL-10-/- MOG + IL-2 + RA vs IL-10-/- Veh by two-way ANOVA and Tukey post hoc test.


Retinal inflammatory scores of mice immunized s . c . with PBS + DMSO ( Veh ) , IRBP + DMSO ( IRBP ) or IRBP + RA + IL - 2 21 and 7 days before induction of EAU by s . c . injection of IRBP emulsified in CFA . Bars are mean + SEM ( n = 6 ) , * p < 0 . 05 and * * * p < 0 . 001 , IRBP + IL - 2 + RA vs Veh , # # # p < 0 . 001 IRBP + IL - 2 + RA vs IRBP by two - way ANOVA with 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

Retinal inflammatory scores of mice immunized s.c. with PBS + DMSO (Veh), IRBP + DMSO (IRBP) or IRBP + RA + IL-2 21 and 7 days before induction of EAU by s.c. injection of IRBP emulsified in CFA. Bars are mean + SEM (n=6), *p<0.05 and ***p<0.001, IRBP + IL-2 + RA vs Veh, ###p<0.001 IRBP + IL-2 + RA vs IRBP by two-way ANOVA with Tukey post hoc test.


Representative fundus images of each group of mice 35 days after induction of EAU . Arrowheads indicate area with retinal vasculitis and the asterisk indicates a region of vitreous haze , where the image appears blurred .

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

Representative fundus images of each group of mice 35 days after induction of EAU. Arrowheads indicate area with retinal vasculitis and the asterisk indicates a region of vitreous haze, where the image appears blurred.


Retinal inflammation scores in HEL - TCR transgenic mice were s . c . injected with PBS + DMSO ( Veh ) or HEL + RA + IL - 2 on pn 18 ( 1x ) or on pn 18 and 25 ( 2x ) . Results are mean + SEM ( n = 3 , 4 or 5 ) . * p < 0 . 05 and * * * p < 0 . 001 , HEL + IL - 2 + RA 1x vs Veh , # # p < 0 . 01 HEL + IL - 2 + RA 2x vs Veh by two - way ANOVA with 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

Retinal inflammation scores in HEL-TCR transgenic mice were s.c. injected with PBS + DMSO (Veh) or HEL + RA + IL-2 on pn 18 (1x) or on pn 18 and 25 (2x). Results are mean + SEM (n=3, 4 or 5). *p<0.05 and ***p<0.001, HEL + IL-2 + RA 1x vs Veh, ##p<0.01 HEL + IL-2 + RA 2x vs Veh by two-way ANOVA with Tukey post hoc test.


Representative photographs of each group of retinal fundus of HEL - TCR transgenic mice at pn 35 . Arrowheads indicate representative areas with retinal vasculitis .

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

Representative photographs of each group of retinal fundus of HEL-TCR transgenic mice at pn 35. Arrowheads indicate representative areas with retinal vasculitis.


E Representative FACS plots of IL - 17 + CD4 or IL - 10 + CD4 T cells in the retina on day 36 of HEL - TCR transgenic mice . Cells were gated on live single CD3 + CD4 + and CD3 + CD4 + LAG - 3 + Foxp3 - respectively .

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

E Representative FACS plots of IL-17+ CD4 or IL-10+ CD4 T cells in the retina on day 36 of HEL-TCR transgenic mice. Cells were gated on live single CD3+CD4+ and CD3+CD4+LAG-3+Foxp3- respectively.


F Mean absolute number of IL - 17 + CD4 T cells in the retina ( upper panels ) or mean percentages of IL - 10 + LAG - 3 + Foxp3 - CD4 T cells in the spleens of of HEL - TCR transgenic mice with vasculitis ( lower panels ) . Results are mean + SEM ( n = 3 , 4 or 5 ) . * * p < 0 . 01 and * * * p < 0 . 001 by one way ANOVA with Bonferroni 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

F Mean absolute number of IL-17+ CD4 T cells in the retina (upper panels) or mean percentages of IL-10+ LAG-3+ Foxp3- CD4 T cells in the spleens of of HEL-TCR transgenic mice with vasculitis (lower panels). Results are mean + SEM (n=3, 4 or 5). **p <0.01 and ***p <0.001 by one way ANOVA with Bonferroni post hoc test.


A Domain architecture of S . pombe bromodomain AAA - ATPases . The D1 and D2 ATPase domains ( AAA ) are shaded blue and orange , respectively , and the bromodomain ( BD ) is grey . The percentage similarity to each other and the indicated proteins was determined by FASTA sequence comparison [ 74 ] using the scoring matrix BLOSUM50 .

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

A Domain architecture of S. pombe bromodomain AAA-ATPases. The D1 and D2 ATPase domains (AAA) are shaded blue and orange, respectively, and the bromodomain (BD) is grey. The percentage similarity to each other and the indicated proteins was determined by FASTA sequence comparison [74] using the scoring matrix BLOSUM50.


B Venn diagrams showing overlap between genes up - regulated ( 1 . 5 fold ) in abo1Δ mutants with genes up - regulated under the indicated condition , along with the significance of the overlaps ( based on hypergeometric distribution ) .

O O O O O O O O O O O O O O O O O O O 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 Venn diagrams showing overlap between genes up-regulated (1.5 fold) in abo1Δ mutants with genes up-regulated under the indicated condition, along with the significance of the overlaps (based on hypergeometric distribution).


A Normalised cumulative nucleosome ( 150 ± 30 bp size class ) position frequency profiles for 4013 S . pombe genes aligned at the transcription start site ( TSS ) plotted from low MNase ( biorep1 ) and high MNase ( biorep 2 ) datasets . P - values ( calculated using a two - tailed unpaired t - test ) for the difference in means between random n = 100 subsets of the frequency values at the points indicated by the dotted lines ( " - 4 " nucleosome and " + 4 " nucleosome ) are shown .

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

A Normalised cumulative nucleosome (150 ± 30 bp size class) position frequency profiles for 4013 S. pombe genes aligned at the transcription start site (TSS) plotted from low MNase (biorep1) and high MNase (biorep 2) datasets. P-values (calculated using a two-tailed unpaired t-test) for the difference in means between random n = 100 subsets of the frequency values at the points indicated by the dotted lines ("-4" nucleosome and "+4" nucleosome) are shown.


B Nucleosome position frequency values for the coding regions of 4013 S . pombe genes were k - means clustered ( k = 9 ) using the abo1Δ data from biorep 1 ( low MNase ) and displayed with positive values coloured yellow and other values coloured blue ( left - hand panel ) . The cluster order was then used to display the equivalent wild type frequency values in the right - hand panel .

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

B Nucleosome position frequency values for the coding regions of 4013 S. pombe genes were k-means clustered (k = 9) using the abo1Δ data from biorep 1 (low MNase) and displayed with positive values coloured yellow and other values coloured blue (left-hand panel). The cluster order was then used to display the equivalent wild type frequency values in the right-hand panel.


C Level of histone gene mRNAs was determined by qRT - PCR . Data are the mean of four independent biological repeats and error bars are ± SEM . Two - tailed unpaired t - tests showed no significant differences ( P > 0 . 05 ) between wild type and abo1Δ cells .

C Level of histone gene mRNAs was determined by qRT-PCR. Data are the mean of four independent biological repeats and error bars are ±SEM. Two-tailed unpaired t-tests showed no significant differences (P > 0.05) between wild type and abo1Δ cells.


D Whole cell extracts were subjected to western blotting with histone H3 ( Abcam ) , histone H2A ( Abcam ) and tubulin antibodies . Examples of the primary data are shown ( left ) along with a quantification of histone H2A and H3 levels normalized to tubulin ( right ) . Data are the mean of at least three independent repeats and error bars represent ± SEM . P - values were calculated using a two - tailed unpaired 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

D Whole cell extracts were subjected to western blotting with histone H3 (Abcam), histone H2A (Abcam) and tubulin antibodies. Examples of the primary data are shown (left) along with a quantification of histone H2A and H3 levels normalized to tubulin (right). Data are the mean of at least three independent repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test.


A Whole cell extracts ( WCE ) were prepared from the indicated strains , immunoprecipitated ( IP ) with anti - GFP antibody and subjected to western blotting with anti - V5 - Pk ( Serotec ) and anti - GFP ( Life Technologies ) antibodies . Data are representative of three independent biological repeats . B As for A .

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

A Whole cell extracts (WCE) were prepared from the indicated strains, immunoprecipitated (IP) with anti-GFP antibody and subjected to western blotting with anti-V5-Pk (Serotec) and anti-GFP (Life Technologies) antibodies. Data are representative of three independent biological repeats.B As for A.


C RNA purified from wild type , abo1Δ and spt16 - 18 cells was analysed by northern blotting using a probe to the 3 ′ end of spbc19c7 . 11 ( top panel ) . RNA ( 5 µg ) used for northern blotting was analysed on an ethidium bromide stained 1 % TAE agarose gel ( bottom panel ) . Data are representative of two independent biological repeats .

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

C RNA purified from wild type, abo1Δ and spt16-18 cells was analysed by northern blotting using a probe to the 3′ end of spbc19c7.11 (top panel). RNA (5 µg) used for northern blotting was analysed on an ethidium bromide stained 1% TAE agarose gel (bottom panel). Data are representative of two independent biological repeats.


D RNA purified from wild type , and abo1Δ cells was analysed by strand specific RT - PCR . RNA from hip1Δ cells was analysed as a control . One primer , complementary to either the forward or reverse transcripts , was included during the reverse transcription step the second primer was then added during PCR amplification . Control reactions omitting the reverse transcription step ( - RT ) were included to demonstrate the absence of contaminating genomic DNA . Data are representative of two independent biological repeats .

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

D RNA purified from wild type, and abo1Δ cells was analysed by strand specific RT-PCR. RNA from hip1Δ cells was analysed as a control. One primer, complementary to either the forward or reverse transcripts, was included during the reverse transcription step the second primer was then added during PCR amplification. Control reactions omitting the reverse transcription step (-RT) were included to demonstrate the absence of contaminating genomic DNA. Data are representative of two independent biological repeats.


E The indicated strains were subjected to ChIP analysis with anti - GFP antibodies and the resulting DNA was analysed by qPCR for the indicated locus . Data are the mean of three independent biological repeats and error bars represent ± SEM . P - values were calculated using a two - tailed unpaired t - test . Significant ( P < 0 . 05 ) values are indicated .

E The indicated strains were subjected to ChIP analysis with anti-GFP antibodies and the resulting DNA was analysed by qPCR for the indicated locus. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test. Significant (P < 0.05) values are indicated.


F ChIP analysis of histone H3 levels at act1 + was determined by qPCR . Data are the mean of three independent biological repeats and error bars represent ± SEM . P - value was calculated using a two - tailed unpaired 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

F ChIP analysis of histone H3 levels at act1+ was determined by qPCR. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-value was calculated using a two-tailed unpaired t-test.


G The indicated strains were subjected to ChIP analysis as described for E .

G The indicated strains were subjected to ChIP analysis as described for E.


H Log phase cells were subjected to five - fold serial dilution and spotted onto rich ( YE5S ) agar or agar supplemented with MMS ( 0 . 006 % ) . Plates were incubated for 3 - 5 days at 30 ° C or 7 days at 20 ° C . Images are representative of three independent biological repeats .

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

H Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with MMS (0.006%). Plates were incubated for 3-5 days at 30°C or 7 days at 20°C. Images are representative of three independent biological repeats.


A The position of ura4 + reporter alleles in centromere 1 is shown in the top panel . Strains containing the imr : : ura4 + allele were grown to log phase in YE5S medium , subjected to 5 - fold serial dilutions and spotted onto YE5S agar or YE5S agar supplemented with 5 - FOA ( 1 mg / ml ) . Data are representative of three independent biological repeats .

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

A The position of ura4+ reporter alleles in centromere 1 is shown in the top panel. Strains containing the imr::ura4+ allele were grown to log phase in YE5S medium, subjected to 5-fold serial dilutions and spotted onto YE5S agar or YE5S agar supplemented with 5-FOA (1 mg/ml). Data are representative of three independent biological repeats.


B Strains carrying the otr : : ura4 + were analysed as described for A .

B Strains carrying the otr::ura4+ were analysed as described for A.


C RNA was purified from the indicated strains and the level of centromeric transcripts determined by qRT - PCR . Data is the mean of three independent repeats and error bars indicate ± SEM . P - value was calculated using a two - tailed unpaired 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

C RNA was purified from the indicated strains and the level of centromeric transcripts determined by qRT-PCR. Data is the mean of three independent repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired t-test.


D RNA was purified from the indicated strains and subjected to qRT - PCR for the subtelomeric gene , tlh1 + . Data is the mean of three biological repeats and error bars indicate ± SEM . P - value was calculated using a two - tailed unpaired Student ' s t - test .

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

D RNA was purified from the indicated strains and subjected to qRT-PCR for the subtelomeric gene, tlh1+. Data is the mean of three biological repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired Student's t-test.


E The position of insertion of ade6 + reporter allele in the silent mat locus is shown in the top panel . Log phase cells containing the mat3 - M : : ade6 + allele were subjected to five - fold serial dilution and spotted onto YE5S agar plates lacking adenine ( Low Ade ) were incubated for 4 days at 30 ° C . Data are representative of at least three independent biological repeats . Note , microarray analysis indicates that deletion of abo1 + does not influence the expression of ade6 + ( or ura4 + ) when these genes are present at their normal genomic loci .

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

E The position of insertion of ade6+ reporter allele in the silent mat locus is shown in the top panel. Log phase cells containing the mat3-M::ade6+ allele were subjected to five-fold serial dilution and spotted onto YE5S agar plates lacking adenine (Low Ade) were incubated for 4 days at 30°C. Data are representative of at least three independent biological repeats. Note, microarray analysis indicates that deletion of abo1+ does not influence the expression of ade6+ (or ura4+) when these genes are present at their normal genomic loci.


A ChIP analysis was performed on wild type ( untagged ) and abo1 - GFP cells and the resulting DNA analysed by qPCR for centromeric ( dh , dg and imr ) repeat sequences . Data are the mean of four independent biological repeats and error bars represent ± SEM . P - values calculated using a two - tailed unpaired t - test indicates that all loci are significantly enriched ( P < 0 . 05 ) relative the untagged control .

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

A ChIP analysis was performed on wild type (untagged) and abo1-GFP cells and the resulting DNA analysed by qPCR for centromeric (dh, dg and imr) repeat sequences. Data are the mean of four independent biological repeats and error bars represent ±SEM. P-values calculated using a two-tailed unpaired t-test indicates that all loci are significantly enriched (P < 0.05) relative the untagged control.


B The indicated strains expressing GFP - Swi6 were subjected to ChIP analysis . The level of centromeric ( dg ) repeat sequences relative to the euchromatic control locus , adh1 + was determined by qPCR and scaled to a clr4Δ ( - H3 K9me control ) mutant . Data are the mean of two independent ChIP experiments and error bars represent the range of the data .

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

B The indicated strains expressing GFP-Swi6 were subjected to ChIP analysis. The level of centromeric (dg) repeat sequences relative to the euchromatic control locus, adh1+ was determined by qPCR and scaled to a clr4Δ (- H3 K9me control) mutant. Data are the mean of two independent ChIP experiments and error bars represent the range of the data.


C ChIP analysis of histone H3 levels at centromeric ( dg ) repeat sequences was determined by qPCR . Data are the mean of three independent ChIP experiments and error bars are ± SEM . P - values were calculated using a two - tailed unpaired t - test .

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

C ChIP analysis of histone H3 levels at centromeric (dg) repeat sequences was determined by qPCR. Data are the mean of three independent ChIP experiments and error bars are ±SEM. P-values were calculated using a two-tailed unpaired t-test.


D . ChIP analysis of Abo1 - GFP at centromeric ( dh and imr ) repeat sequences regions in wild type and pob3Δ cells . Data are the mean of three independent experiments and error bars represent ± SEM . P - values , calculated using a two - tailed unpaired t - test , indicated no significant difference ( P > 0 . 05 ) for wild type and pob3Δ cells .

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

D. ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences regions in wild type and pob3Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant difference (P > 0.05) for wild type and pob3Δ cells.


E ChIP analysis of Abo1 - GFP at centromeric ( dh and imr ) repeat sequences in wild type and pob3Δ cells . Data are the mean of duplicate experiments and error bars represent the range of the data .

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

E ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences in wild type and pob3Δ cells. Data are the mean of duplicate experiments and error bars represent the range of the data.


F ChIP analysis of Abo1 - GFP over the dh and imr regions in wild type and swi6Δ cells . Data are the mean of three independent experiments and error bars represent ± SEM . P - values , calculated using a two - tailed unpaired t - test , indicated no significant differences ( P > 0 . 05 ) for wild type and swi6Δ cells .

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

F ChIP analysis of Abo1-GFP over the dh and imr regions in wild type and swi6Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant differences (P > 0.05) for wild type and swi6Δ cells.


A RNA was extracted from mid log phase cells and Tf2 mRNA levels were determined by qRT - PCR , normalised to act1 + mRNA and scaled relative to the wild type level . Data are the mean of three independent biological repeats and error bars represent ± SEM .

A RNA was extracted from mid log phase cells and Tf2 mRNA levels were determined by qRT-PCR, normalised to act1+ mRNA and scaled relative to the wild type level. Data are the mean of three independent biological repeats and error bars represent ±SEM.


B Mid log phase cells with the indicated integrated lacZ reporter were subjected to quantitative β - galactosidase assays . Data are the mean of three independent biological repeats and error bars represent ± SEM . C As for

O O O O O O O O O O O O O O O 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 Mid log phase cells with the indicated integrated lacZ reporter were subjected to quantitative β-galactosidase assays. Data are the mean of three independent biological repeats and error bars represent ±SEM.C As for B


D ChIP DNA samples from wild type ( untagged ) and abo1 - GFP cells were analysed by qPCR for Tf2 LTR . Data are the mean of four independent biological repeats and error bars represent ± SEM .

D ChIP DNA samples from wild type (untagged) and abo1-GFP cells were analysed by qPCR for Tf2 LTR. Data are the mean of four independent biological repeats and error bars represent ±SEM.


E Normalised cumulative nucleosome ( 150 ± 30 bp ) position frequency profiles for Tf2 LTR retrotransposons aligned at the ATG plotted from low MNase ( biorep1 ) and high MNase ( biorep 2 ) datasets . P - values were calculated using a two - tailed unpaired 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

E Normalised cumulative nucleosome (150 ± 30 bp) position frequency profiles for Tf2 LTR retrotransposons aligned at the ATG plotted from low MNase (biorep1) and high MNase (biorep 2) datasets.P-values were calculated using a two-tailed unpaired t-test.


A Log phase cells were subjected to five - fold serial dilution and spotted onto rich ( YE5S ) agar or agar supplemented with TBZ ( 15 μg / ml ) and incubated for 3 - 5 days 30 ° C . Data are representative of at three independent biological repeats .

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

A Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with TBZ (15 μg/ml) and incubated for 3-5 days 30°C. Data are representative of at three independent biological repeats.