( b ) GFP - LC3 - expressing RPE were depleted of myosin VI by siRNA . were labelled with Texas - red - dextran ( red ) for 4 h , before chase into fresh medium containing 1 μM MG132 for 2 h . were processed for immunofluorescence microscopy immunostained for GFP ( green ) . Nuclei were labelled with Hoechst ( blue ) . insets represent higher magnifications of areas outlined in main panels . ( c ) Mander ' s overlap coefficients for degree of LC3 signal co - localizing with dextran were calculated using Volocity software . results represent > 20 from n = 3 independent experiments are illustrated as a box whisker plot . box represents median , 25th 75th percentiles whiskers represent maximum minimum .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP-LC3-expressing RPE cells were depleted of myosin VI by siRNA. Cells were pulse labelled with Texas-red-dextran (red) for 4 h, before chase into fresh medium containing 1 μM MG132 for 2 h. Cells were processed for immunofluorescence microscopy and immunostained for GFP (green). Nuclei were labelled with Hoechst (blue). The insets represent higher magnifications of the areas outlined in the main panels. (c) Mander's overlap coefficients for the degree of LC3 signal co-localizing with dextran were calculated using Volocity software. The results represent >20 cells from n = 3 independent experiments and are illustrated as a box and whisker plot. The box represents the median, 25th and 75th percentiles and whiskers represent the maximum and minimum.


( d ) RPE stably expressing GFP - LC3 were depleted of myosin VI Tom1 by siRNA . were - labelled with Texas - red ( TR ) - dextran for 16 h followed by a chase period of 4 h . were processed for immunoelectron microscopy labelled with 15 nm gold particles against GFP - LC3 5 nm gold particles against Texas - red - dextran . Scale bars , 20 μm ( a , b ) ; 200 nm ( d ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RPE cells stably expressing GFP-LC3 were depleted of myosin VI and Tom1 by siRNA. Cells were pulse-labelled with Texas-red (TR)-dextran for 16 h followed by a chase period of 4 h. Cells were processed for immunoelectron microscopy and labelled with 15 nm gold particles against GFP-LC3 and 5 nm gold particles against Texas-red-dextran. Scale bars, 20 μm (a,b); 200 nm (d).


Representative immunofluorescence of cilium in WT or Mecp2 null MEF . were starved for 48 h before with anti - γ - tubulin ( green ) anti - acetylated α - tubulin ( red ) antibodies . Merge of channels DAPI ( blue ) is depicted for both genotypes . Scale bar 20 µm , 10 µm in enlarged image . Arrows indicate not ciliated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunofluorescence of primary cilium in WT or Mecp2 null MEF cells. Cells were starved for 48 h before staining with anti-γ-tubulin (green) and anti-acetylated α-tubulin (red) antibodies. Merge of all channels and DAPI staining (blue) is depicted for both genotypes. Scale bar 20 µm, and 10 µm in the enlarged image. Arrows indicate not ciliated cells.


Cilia were counted analysed with ImageJ to quantify of ciliated ( n = 134 WT n = 140 Mecp2 null ) ( B ) cilium ( n = 104 WT n = 43 Mecp2 null ) ( C ) . graphs show average of three independent experiments ( mean ± S . E . , * * p < 0 . 01 , by Mann - Whitney ; * * * p < 0 . 001 , by Student ' s t - ) .

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

Cilia were counted and analysed with ImageJ to quantify the percentage of ciliated cells (n=134 WT and n=140 Mecp2 null cells) (B) and the cilium length (n=104 WT and n=43 Mecp2 null cells) (C). The graphs show the average of three independent experiments (mean ± S.E., **p<0.01, by Mann-Whitney test; ***p<0.001, by Student's t-test).


Representative immunofluorescence of cilium in hTERT - RPE - 1 silenced with MECP2 - or control - siRNAs . were starved for 48 h before with anti - γ - tubulin ( green ) anti - acetylated α - tubulin ( red ) antibodies . Scale bar 10 µm , 5 µm in enlarged image . Arrows indicate not ciliated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunofluorescence of primary cilium in hTERT-RPE-1 cells silenced with MECP2- or control-siRNAs. Cells were starved for 48 h before staining with anti-γ-tubulin (green) and anti-acetylated α-tubulin (red) antibodies. Scale bar 10 µm, and 5 µm in the enlarged image. Arrows indicate not ciliated cells.


Representative Western blotting showing MeCP2 expression in CTRL silenced hTERT - RPE - 1 , with respect to GAPDH , used as loading control .

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Representative Western blotting showing the MeCP2 expression in CTRL and silenced hTERT-RPE-1 cells, with respect to GAPDH, used as loading control.


of MeCP2 positive was calculated by analysing n = 1000 CTRL silenced hTERT - RPE - 1 ( mean ± S . E . , * * * p < 0 . 001 , by Student ' s t ) .

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

The percentage of MeCP2 positive cells was calculated by analysing n=1000 CTRL and silenced hTERT-RPE-1 cells (mean ± S.E., ***p<0.001, by Student's t test).


Cilia were counted analysed with ImageJ to quantify of ciliated ( n = 191 CTRL n = 197 siMeCP2 ) ( G ) cilium ( n = 50 CTRL n = 50 siMeCP2 ) ( H ) . graphs show mean ± S . E of three independent experiments ( * * * p < 0 . 001 , by Mann - Whitney ( G ) or Student ' s t - ( H ) ) .

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

Cilia were counted and analysed with ImageJ to quantify the percentage of ciliated cells (n=191 CTRL and n=197 siMeCP2 cells) (G) and the cilium length (n=50 CTRL and n=50 siMeCP2 cells) (H). The graphs show the mean ± S.E of three independent experiments (***p<0.001, by Mann-Whitney test (G) or Student's t-test (H)).


Representative immunofluorescence of cilium in WT Mecp2 null cortical neurons at DIV7 DIV14 . Scale bar 10 µm , 5 µm in enlarged image . Arrows indicate not ciliated .

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Representative immunofluorescence of primary cilium in WT and Mecp2 null primary cortical neurons at DIV7 and DIV14. Scale bar 10 µm, and 5 µm in the enlarged image. Arrows indicate not ciliated cells.


Neurons were fixed at DIV3 , DIV7 DIV14 cilium detected by immunostaining with anti - AC3 antibody . of ciliated ( J ) cilium ( K ) were analysed . Histograms represent average of three independent experiments , analysing neurons derived from 3 different experiments ( n = 156 WT DIV3 ; n = 49 KO DIV3 ; n = 177 WT DIV7 ; n = 82 KO DIV7 ; n = 59 WT DIV14 ; n = 37 KO DIV14 ) ( mean ± S . E . , * * p < 0 . 01 , * * * p < 0 . 001 , by two - way ANOVA followed by Bonferroni post hoc ) .

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

Neurons were fixed at DIV3, DIV7 and DIV14 and primary cilium detected by immunostaining with anti-AC3 antibody. Percentage of ciliated cells (J) and cilium length (K) were analysed. Histograms represent the average of three independent experiments, analysing neurons derived from 3 different experiments (n=156 WT DIV3; n=49 KO DIV3; n=177 WT DIV7; n=82 KO DIV7; n=59 WT DIV14; n=37 KO DIV14) (mean ± S.E., **p<0.01, ***p<0.001, by two-way ANOVA followed by Bonferroni post hoc test).


cortical astrocytes were fixed at DIV15 stained with anti - acetylated α - tubulin ( red ) anti - ɣ - tubulin ( green ) antibodies . Scale bar 20 µm , 10 µm in enlarged image . Arrow indicates not ciliated .

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Primary cortical astrocytes were fixed at DIV15 and stained with anti-acetylated α-tubulin (red) and anti-ɣ-tubulin (green) antibodies. Scale bar 20 µm, and 10 µm in the enlarged image. Arrow indicates not ciliated cell.


of ciliated ( M ) cilium ( N ) were analysed ( n = 26 WT n = 34 Mecp2 null astrocytes ) . Histograms represent average of three independent experiments ( mean ± S . E . , * p < 0 . 05 , Student ' s t ) .

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

Percentage of ciliated cells (M) and cilium length (N) were analysed (n=26 WT and n=34 Mecp2 null astrocytes). Histograms represent the average of three independent experiments (mean ± S.E., *p<0.05, Student's t test).


Representative immunofluorescence of Mecp2 null cortical neurons infected at DIV0 with lentiviruses expressing GFP or MeCP2iresGFP . Neurons were fixed at DIV7 immunostained with anti - AC3 antibody to detect cilium . GFP - positive represent infected . Scale bar 20 µm , 10 µm in enlarged image .

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Representative immunofluorescence of Mecp2 null cortical neurons infected at DIV0 with lentiviruses expressing GFP or MeCP2iresGFP. Neurons were fixed at DIV7 and immunostained with anti-AC3 antibody to detect the primary cilium. GFP-positive cells represent infected cells. Scale bar 20 µm, and 10 µm in the enlarged image.


Western blot comparing MeCP2 in Mecp2 null neurons infected with lentiviruses expressing GFP or MeCP2iresGFP with respect to WT neurons . Data are normalized to total content , visualized by a TGX stain - free technology .

O O O O O O O O O O O O O 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 comparing MeCP2 levels in Mecp2 null neurons infected with lentiviruses expressing GFP or MeCP2iresGFP with respect to WT neurons. Data are normalized to total protein content, visualized by a TGX stain-free technology.


MECP2 expression in null neurons induced a significant recovery of ciliogenesis . ( C ) graph represents of ciliated , counting n = 50 WT n = 100 null of two different preparations ( mean ± S . E . , * * * p < 0 . 001 , by two - way ANOVA followed by Bonferroni post - hoc ) . Statistical reported a significant interaction ( F ( 1 , 135 ) = 8 . 264 ) , a significant genotype effect ( F ( 1 , 135 ) = 37 . 59 ) a significant infection effect ( F ( 1 , 135 ) = 26 . 89 ) . ( D ) graph represents mean ± S . E . of cilium in WT Mecp2 null neurons infected with GFP or MeCP2iresGFP expressing lentiviruses , counting n = 28 of two different preparations ( * * p < 0 . 01 , * * * p < 0 . 001 , by two - way ANOVA followed by Bonferroni post - hoc ) . Two - way ANOVA reported a significant interaction ( F ( 1 , 103 ) = 6 . 405 ) , a significant genotype effect ( F ( 1 , 103 ) = 35 ) a significant infection effect ( F ( 1 . 103 ) = 4 . 039 ) .

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

MECP2 expression in null neurons induced a significant recovery of ciliogenesis. (C) The graph represents the percentage of ciliated cells, counting n=50 WT and n=100 null cells of two different preparations (mean ± S.E., ***p<0.001, by two-way ANOVA followed by Bonferroni post-hoc test). Statistical analysis reported a significant interaction (F (1,135) = 8.264), a significant genotype effect (F (1,135) = 37.59) and a significant infection effect (F (1,135) = 26.89). (D) The graph represents the mean ± S.E. of cilium length in WT and Mecp2 null neurons infected with GFP or MeCP2iresGFP expressing lentiviruses, counting n=28 cells of two different preparations (**p<0.01, ***p<0.001, by two-way ANOVA followed by Bonferroni post-hoc test). Two-way ANOVA reported a significant interaction (F (1,103) = 6.405), a significant genotype effect (F (1,103) = 35) and a significant infection effect (F (1.103) = 4.039).


MECP2 - silenced or control hTERT - RPE - 1 were starved for 48 h before treatment with 200 nM 500 nM SAG for 24 h . were stained with anti - Smo anti - acetylated α - tubulin antibodies . percentages of Smo - positive cilia were calculated . graph shows average of three independent experiments ( mean ± S . E . , * * * p < 0 . 001 , by two - way ANOVA followed by Bonferroni post hoc ) . Two - way ANOVA demonstrated a significant silencing effect ( F ( 1 , 12 ) = 160 ; p < 0 . 0001 ) , a significant SAG treatment ( F ( 2 , 12 ) = 259 . 4 ; p < 0 . 0001 ) a significant interaction ( F ( 2 , 12 ) = 36 . 85 ; p < 0 . 0001 ) .

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

MECP2-silenced or control hTERT-RPE-1 cells were starved for 48 h before treatment with 200 nM and 500 nM SAG for 24 h. Cells were stained with anti-Smo and anti-acetylated α-tubulin antibodies. The percentages of Smo-positive cilia were calculated. The graph shows the average of three independent experiments (mean ± S.E., *** p<0.001, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA demonstrated a significant silencing effect (F (1, 12) = 160; p<0.0001), a significant SAG treatment (F (2, 12) = 259.4; p<0.0001) and a significant interaction (F (2, 12) = 36.85; p<0.0001).


WT or Mecp2 null MEF were treated with SAG ( 200 500 nM ) for 24 h percentages of Smo - positive cilia were calculated . graph shows average of three independent experiments ( mean ± S . E . , * p < 0 . 05 , by two - way ANOVA followed by Bonferroni post hoc ) . Two - way ANOVA indicated a significant genotype effect ( F ( 1 , 12 ) = 11 . 22 ; p < 0 . 01 ) a significant SAG treatment ( F ( 2 , 12 ) = 11 . 7 ; p < 0 . 0001 ) .

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

WT or Mecp2 null MEF cells were treated with SAG (200 and 500 nM) for 24 h and the percentages of Smo-positive cilia were calculated. The graph shows the average of three independent experiments (mean ± S.E., *p<0.05, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA indicated a significant genotype effect (F (1, 12) = 11.22; p<0.01) and a significant SAG treatment (F (2, 12) = 11.7; p<0.0001).


Gli1 were measured by Western blot in untreated ( UT ) 100 nM SAG - treated WT Mecp2 null MEF ( n = 5 / 6 ) . Representative bands of Gli1 , Mecp2 α - tubulin are depicted above histogram , which shows mean ± S . E of of Gli1 expression , compared to untreated WT samples ( * * p < 0 . 01 , * * * p < 0 . 001 , by two - way ANOVA , followed by Bonferroni post hoc ) . Two - way ANOVA reported a significant interaction ( F ( 1 , 18 ) = 6 . 652 ; p < 0 . 05 ) , a significant genotype effect ( F ( 1 , 18 ) = 15 . 95 ; p < 0 . 001 ) a significant treatment ( F ( 1 , 18 ) = 20 . 15 ; p < 0 . 001 ) .

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

Gli1 protein levels were measured by Western blot in untreated (UT) and 100 nM SAG-treated WT and Mecp2 null MEF cells (n=5/6). Representative bands of Gli1, Mecp2 and α-tubulin are depicted above the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to the untreated WT samples (**p<0.01, ***p<0.001, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA reported a significant interaction (F (1, 18) = 6.652; p<0.05), a significant genotype effect (F (1,18) = 15.95; p<0.001) and a significant treatment (F (1, 18) = 20.15; p<0.001).


Gli1 were measured by Western blot in WT Mecp2 null neurons , following SAG treatment ( 200 400 nM ) . Representative bands of Gli1 , Mecp2 α - tubulin are depicted above histogram , which shows mean ± S . E of of Gli1 expression , compared to untreated WT samples ( * p < 0 . 05 , * * * p < 0 . 001 , by two - way ANOVA , followed by Bonferroni post hoc ) . Two - way ANOVA indicated a significant interaction ( F ( 2 , 20 ) = 3 . 788 ; p < 0 . 05 ) , a significant genotype effect ( F ( 1 , 20 ) = 4 . 362 ; p < 0 . 05 ) a significant treatment ( F ( 2 , 20 ) = 13 . 5 ; p < 0 . 001 ) .

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

Gli1 protein levels were measured by Western blot in WT and Mecp2 null neurons, following SAG treatment (200 and 400 nM). Representative bands of Gli1, Mecp2 and α-tubulin are depicted above the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to the untreated WT samples (*p<0.05, ***p<0.001, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA indicated a significant interaction (F (2, 20) = 3.788; p<0.05), a significant genotype effect (F (1, 20) = 4.362; p<0.05) and a significant treatment (F (2, 20) = 13.5; p<0.001).


Gli1 mRNA expression upon SAG exposure in MEF ( n = 7 WT n = 9 null samples ) ( F ; SAG 100 nM ) cortical neurons ( n = 15 WT n = 12 null samples ) ( G ; SAG 400 nM ) . graphs show mean ± S . E . of fold change derived from at least three independent experiments ( mean ± S . E . , * * p < 0 . 01 , by two - way ANOVA , followed by Bonferroni post hoc ) .

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Gli1 mRNA expression levels upon SAG exposure in MEF cells (n=7 WT and n=9 null samples) (F; SAG 100 nM) and cortical neurons (n= 15 WT and n=12 null samples) (G; SAG 400 nM). The graphs show the mean ± S.E. of the fold change derived from at least three independent experiments (mean ± S.E., **p<0.01, by two-way ANOVA, followed by Bonferroni post hoc test).


Representative immunostaining of Arl13b ( red ) γ - tubulin ( green ) in P14 mouse cortical layers . Nuclei were stained with DAPI ( blue ) . Scale bar 10 µm .

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Representative immunostaining of Arl13b (red) and γ-tubulin (green) in the P14 mouse cortical layers. Nuclei were stained with DAPI (blue). Scale bar 10 µm.


cilium was measured in cortex of Mecp2 null mice Average cilium was measured in cortex ( mean ± S . E . , n = 4 WT n = 3 Mecp2 null ) ( B ; * p < 0 . 05 , by Mann - Whitney ) for each cortical layer ( C ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , by two - way ANOVA followed by Bonferroni post hoc ) . In C , cilium is analysed in separate cortical layers while in B , values indicate average of cilium lengths for each layer .

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Primary cilium length was measured in the cortex of Mecp2 null mice Average cilium length was measured in the cortex (mean ± S.E., n=4 WT and n=3 Mecp2 null) (B; *p<0.05, by Mann-Whitney test) and for each cortical layer (C; ***p<0.001, **p<0.01, *p<0.05, by two-way ANOVA followed by Bonferroni post hoc test). In C, cilium length is analysed in separate cortical layers while in B, the values indicate the average of cilium lengths for each layer.


Representative immunostaining of Arl13b ( green ) in internal ( IGL ) external ( EGL ) granular layers of Mecp2 null ( n = 3 ) heterozygous ( het ) cerebella ( n = 3 ) at P7 , corresponding WT ( n = 3 n = 3 ) . Nuclei were stained with DAPI ( blue ) . Scale bar 5 µm .

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

Representative immunostaining of Arl13b (green) in the internal (IGL) and external (EGL) granular layers of Mecp2 null (n=3) and heterozygous (het) cerebella (n=3) at P7, and the corresponding WT (n=3 males and n=3 females). Nuclei were stained with DAPI (blue). Scale bar 5 µm.


cilium was measured in cerebellum of Mecp2 null heterozygous mice . Histograms show mean ± S . E . of cilium ( n = 3 WT , n = 3 WT , n = 3 Mecp2 null Mecp2 het ; * * p < 0 . 01 , * * * p < 0 . 001 , by Mann - Whitney ) in Mecp2 null ( E ) heterozygous ( F ) mice , compared to corresponding WT .

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

Primary cilium length was measured in the cerebellum of Mecp2 null and heterozygous mice. Histograms show the mean ± S.E. of cilium length (n=3 males WT, n=3 females WT, n=3 Mecp2 null and Mecp2 het; **p<0.01, ***p<0.001, by Mann-Whitney test) in Mecp2 null (E) and heterozygous (F) mice, compared to the corresponding WT.


Gli1 were measured by Western blot in WT Mecp2 null cerebellum ( n = 7 WT n = 6 Mecp2 null ) . Representative bands of Gli1 α - tubulin are depicted beside histogram , which shows mean ± S . E of of Gli1 expression , compared to WT ( * p < 0 . 05 , by Student ' s t ) .

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

Gli1 protein levels were measured by Western blot in WT and Mecp2 null cerebellum (n=7 WT and n=6 Mecp2 null). Representative bands of Gli1 and α-tubulin are depicted beside the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to WT animals (*p<0.05, by Student's t test).


mRNA expression of Gli1 its downstream genes ( Cyclin D1 D2 ) were analyzed in WT Mecp2 null cerebella ( n = 8 WT n = 7 Mecp2 null ) . graph shows fold change of transcripts compared to WT ( mean ± S . E . , * p < 0 . 05 , by Student ' s t ) .

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

The mRNA expression levels of Gli1 and its downstream genes (Cyclin D1 and D2) were analyzed in WT and Mecp2 null cerebella (n=8 WT and n=7 Mecp2 null). The graph shows the fold change of transcripts compared to WT (mean ± S.E., *p<0.05, by Student's t test).


WT Mecp2 null MEF were starved for 48 h then treated with HDAC6 inhibitor tubacin ( TUB ) ( 1 μM ) for 48 h or with DMSO ( VEH ) or left untreated ( UT ) . were fixed , stained for anti - acetylated α - tubulin cilia were counted . graph shows of ciliated out of three independent experiments ( mean ± S . E . , * p < 0 . 05 , * * p < 0 . 01 , by two - way ANOVA followed by Bonferroni post hoc ) . Two - way ANOVA reported a significant genotype effect ( F ( 1 , 10 ) = 17 . 94 ; p < 0 . 01 ) , a significant tubacin treatment ( F ( 2 , 10 ) = 4 . 425 ; p < 0 . 05 ) a significant interaction ( F ( 2 , 10 ) = 7 . 266 ; p < 0 . 05 ) .

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 O O O 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

WT and Mecp2 null MEF cells were starved for 48 h and then treated with the HDAC6 inhibitor tubacin (TUB) (1 μM) for 48 h or with DMSO (VEH) or left untreated (UT). Cells were fixed, stained for anti-acetylated α-tubulin and cilia were counted. The graph shows the percentage of ciliated cells out of three independent experiments (mean ± S.E., *p<0.05, **p<0.01, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA reported a significant genotype effect (F(1, 10) = 17.94; p<0.01), a significant tubacin treatment (F(2, 10) = 4.425; p<0.05) and a significant interaction (F(2, 10) = 7.266; p<0.05).


cortical neurons were treated at DIV5 with DMSO ( VEH ) or tubacin ( TUB , 1 μM ) for 48 h , or left untreated ( UT ) , fixed at DIV7 stained with anti - AC3 antibody . graph shows of ciliated obtained from three independent experiments counting n = 300 UT , n = 216 VEH treated , n = 267 TUB treated for each genotype ( mean ± S . E . , * p < 0 . 05 , * * p < 0 . 01 , by two - way ANOVA , followed by Bonferroni post hoc ) . Two - way ANOVA reported a significant genotype effect ( F ( 1 , 15 ) = 23 . 42 ; p < 0 . 001 ) , a significant treatment effect ( F ( 2 , 15 ) = 11 . 25 ; p < 0 . 01 ) a significant interaction ( F ( 2 , 15 ) = 4 . 853 ; p < 0 . 05 ) .

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

Primary cortical neurons were treated at DIV5 with DMSO (VEH) or tubacin (TUB, 1 μM) for 48 h, or left untreated (UT), fixed at DIV7 and stained with anti-AC3 antibody. The graph shows the percentage of ciliated cells obtained from three independent experiments counting n=300 UT cells, n=216 VEH treated cells, n=267 TUB treated cells for each genotype (mean ± S.E., *p<0.05, **p<0.01, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA reported a significant genotype effect (F (1, 15) = 23.42; p<0.001), a significant treatment effect (F (2, 15) = 11.25; p<0.01) and a significant interaction (F (2, 15) = 4.853; p<0.05).


MECP2 silenced or control hTERT - RPE - 1 were starved for 48 h , treated with tubacin ( TUB , 1 µM ) for 24 h then exposed to 200 nM SAG for 24 h in presence of tubacin . were stained with anti - Smo anti - acetylated α - tubulin antibodies percentages of Smo - positive cilia were calculated . graph shows averages of three independent experiments ( mean ± S . E . , * * p < 0 . 01 , by three - way ANOVA followed by Tukey ' s post hoc ) . Statistical reported a significant difference between genotype ( F ( 1 , 2 ) = 21 . 47 ; p < 0 . 001 ) , a significant SAG effect ( F ( 2 , 2 ) = 154 . 5 ; p < 0 . 0001 ) a significant tubacin effect ( F ( 1 , 2 ) = 25 . 18 ; p < 0 . 001 ) . In details , a significant interaction between genotype SAG treatment ( F ( 2 , 2 ) = 5 . 413 ; p < 0 . 05 ) between SAG tubacin treatment ( F ( 2 , 2 ) = 6 . 939 ; p < 0 . 01 ) were reported , demonstrating respectively a difference between CTRL MeCP2 depleted in response to SAG a different SAG effect when were treated with tubacin .

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

MECP2 silenced or control hTERT-RPE-1 cells were starved for 48 h, treated with tubacin (TUB, 1 µM) for 24 h and then exposed to 200 nM SAG for 24 h in the presence of tubacin. Cells were stained with anti-Smo and anti-acetylated α-tubulin antibodies and the percentages of Smo-positive cilia were calculated. The graph shows the averages of three independent experiments (mean ± S.E., **p<0.01, by three-way ANOVA followed by Tukey's post hoc test). Statistical analysis reported a significant difference between genotype (F (1, 2) = 21.47; p<0.001), a significant SAG effect (F (2, 2) = 154.5; p<0.0001) and a significant tubacin effect (F (1, 2) = 25.18; p<0.001). In details, a significant interaction between genotype and SAG treatment (F (2, 2) = 5.413; p<0.05) and between SAG and tubacin treatment (F (2, 2) = 6.939; p<0.01) were reported, demonstrating respectively a difference between CTRL and MeCP2 depleted cells in response to SAG and a different SAG effect when cells were treated with tubacin.


Representative immunostaining of Smo ( green ) acetylated α - tubulin ( red ) in control ( CTRL ) silenced ( siMeCP2 ) hTERT - RPE - 1 , after stimulation with 200 nM SAG in presence or absence of HDAC6 inhibitor tubacin ( TUB ) . Scale bar 50 µm , 20 µm in enlarged image .

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

Representative immunostaining of Smo (green) and acetylated α-tubulin (red) in control (CTRL) and silenced (siMeCP2) hTERT-RPE-1 cells, after stimulation with 200 nM SAG in the presence or absence of the HDAC6 inhibitor tubacin (TUB). Scale bar 50 µm, and 20 µm in the enlarged image.


Representative of MAP2 positive WT Mecp2 null neurons ( DIV7 ) . At DIV5 neurons were treated with tubacin ( TUB , 1µM for 48 h ) , Aurora A inhibitor TC - S 7010 ( TC - S , 7 nM for 48 h ) or left untreated morphologically analyzed by Sholl plug - in . Scale bar 20 µm . graph reports mean ± S . E . of number of intersections measured 85 to 145 nm from soma calculated by Sholl for WT ( n = 39 neurons ) Mecp2 null neurons , when left untreated ( n = 57 neurons ) or treated with tubacin ( n = 53 neurons ) or TC - S 7010 ( n = 38 neurons ) . Data from WT null neurons were compared by Mann - Whitney ( * * p < 0 . 01 ) ; drug effects on null were analyzed by one - way ANOVA followed by Dunn ' s post hoc ( * p < 0 . 05 , * * p < 0 . 01 ) .

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

Representative images of MAP2 positive WT and Mecp2 null neurons (DIV7). At DIV5 neurons were treated with tubacin (TUB, 1µM for 48 h), Aurora A inhibitor TC-S 7010 (TC-S, 7 nM for 48 h) or left untreated and morphologically analyzed by Sholl analysis plug-in. Scale bar 20 µm. The graph reports the mean ± S.E. of the number of intersections measured 85 to 145 nm from the soma calculated by Sholl analysis for WT (n=39 neurons) and Mecp2 null neurons, when left untreated (n=57 neurons) or treated with tubacin (n=53 neurons) or TC-S 7010 (n=38 neurons). Data from WT and null neurons were compared by Mann-Whitney test (**p<0.01); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05, **p<0.01).


Representative of WT Mecp2 null neurons ( DIV14 ) immunostained for MAP2 ( ) , Synapsin1 / 2 ( green ) Shank2 ( red ) . Mecp2 null were treated at DIV12 with tubacin ( TUB , 1µM for 48 h ) or Aurora A inhibitor TC - S 7010 ( TC - S , 7 nM for 48 h ) . Scale bar 15 µm , 5 µm in enlarged image .

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

Representative images of WT and Mecp2 null neurons (DIV14) immunostained for MAP2 (white), Synapsin1/2 (green) and Shank2 (red). Mecp2 null cells were treated at DIV12 with tubacin (TUB, 1µM for 48 h) or Aurora A inhibitor TC-S 7010 (TC-S, 7 nM for 48 h). Scale bar 15 µm, and 5 µm in the enlarged image.


graphs represent mean ± S . E . of Synapsin1 / 2 Shank2 density in WT ( n = 67 neurons ) Mecp2 null neurons , when untreated ( n = 51 neurons ) or treated with tubacin ( n = 59 neurons ) or TC - S 7010 ( n = 55 neurons ) . Neurons derived from at least 6 different biological replicates . Data from WT null neurons were compared by Mann - Whitney ( * * p < 0 . 01 , * * * p < 0 . 001 ) ; drug effects on null were analyzed by one - way ANOVA followed by Dunn ' s post hoc ( * p < 0 . 05 , * * * p < 0 . 00 1 ) . graph depicts mean ± S . E . of number of co - localized pre - post - synaptic ( n = 20 neurons deriving from 3 different biological samples ) . Data from WT null neurons were compared by Mann - Whitney ( * * * p < 0 . 001 ) ; drug effects on null were analyzed by one - way ANOVA followed by Dunn ' s post hoc ( * p < 0 . 05 ) .

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

The graphs represent the mean ± S.E. of the Synapsin1/2 and the Shank2 puncta density in WT (n=67 neurons) and Mecp2 null neurons, when untreated (n=51 neurons) or treated with tubacin (n=59 neurons) or TC-S 7010 (n=55 neurons). Neurons derived from at least 6 different biological replicates. Data from WT and null neurons were compared by Mann-Whitney test (**p<0.01, ***p<0.001); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05, ***p<0.001). The graph depicts the mean ± S.E. of the number of co-localized pre- and post-synaptic puncta (n=20 neurons deriving from 3 different biological samples). Data from WT and null neurons were compared by Mann-Whitney test (***p<0.001); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05).


Representative immunostaining of cilia MeCP2 in cultured human fibroblasts from healthy controls ( CTRL , n = 3 ) RTT . Fibroblasts were starved for 24 h then stained for acetylated α - tubulin ( green ) , MeCP2 ( red ) DAPI ( blue ) . Scale bar 50 µm .

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

Representative immunostaining of primary cilia and MeCP2 in cultured human fibroblasts from healthy controls (CTRL, n=3) and RTT patients. Fibroblasts were starved for 24 h and then stained for acetylated α-tubulin (green), MeCP2 (red) and DAPI (blue). Scale bar 50 µm.


Histograms indicate ( mean ± S . E . ) of MeCP2 positive

Histograms indicate (mean ± S.E.) the percentage of MeCP2 positive cells


Histograms indicate ( mean ± S . E . ) of ciliated in control RTT

Histograms indicate (mean ± S.E.) the percentage of ciliated cells in control and RTT fibroblasts


effect of tubacin ( TUB ) treatment ( 250 nM , 24 h ) on cilium of controls ( CTRL ) RTT fibroblasts is reported . graph indicates mean ± S . E of cilium analyzed in untreated ( n = 130 CTRL ; n = 73 705delG ; n = 57 Q244X ; n = 68 R255X ) treated ( n = 25 CTRL ; n = 26 705delG ; n = 26 Q244X ; n = 49 R255X ) ( * * * p < 0 . 001 vs untreated CTRL , § p < 0 . 05 vs corresponding mutated untreated fibroblasts by two - way ANOVA , followed by Bonferroni post hoc ) . Statistical reported a significant treatment effect ( F ( 1 , 429 ) = 26 . 56 ; p < 0 . 0001 ) a significant genotype ( F ( 3 , 429 ) = 42 . 85 ; p < 0 . 001 ) .

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

The effect of tubacin (TUB) treatment (250 nM, 24 h) on primary cilium length of controls (CTRL) and RTT fibroblasts is reported. The graph indicates the mean ± S.E of the cilium length analyzed in untreated (n=130 CTRL; n=73 705delG; n=57 Q244X; n=68 R255X) and treated cells (n=25 CTRL; n=26 705delG; n=26 Q244X; n=49 R255X) (***p<0.001 vs untreated CTRL, §p<0.05 vs corresponding mutated untreated fibroblasts by two-way ANOVA, followed by Bonferroni post hoc test). Statistical analysis reported a significant treatment effect (F (1,429) = 26.56; p<0.0001) and a significant genotype (F(3,429) = 42.85; p<0.001).


A ) Representative spreads of wild - type pachytene Rec8 − / − pachytene - like spermatocytes . spreads were immunostained for SYCP3 ACA . Magnified views are indicated by dashed areas . Schematic representation on magnified chromosomes represents tightly associated sister - AEs ( separated for easier representation ) in paired wild - type homologs , appearance of two distinguishable / separated sister - AEs once REC8 - mediated cohesion is lost in Rec8 − / − univalents . Bars , 10 μm in spreads 1 μm on insets .

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

A) Representative nuclear spreads of wild-type pachytene and Rec8−/− pachytene-like spermatocytes. Nuclear spreads were immunostained for SYCP3 and ACA. Magnified views are indicated by dashed areas. Schematic representation on magnified chromosomes represents tightly associated sister-AEs (separated for easier representation) in paired wild-type homologs, and the appearance of two distinguishable/separated sister-AEs once REC8-mediated cohesion is lost in Rec8−/− univalents. Bars, 10 μm in spreads and 1 μm on insets.


B ) Representative spread of zygotene - like Stag3 mutant spermatocytes . spreads were immunostained for SYCP3 ACA . Magnified views are indicated by dashed areas . Schematic representation on magnified univalents represents : separation of sister - AEs ( centre ) close association of sister - AEs ( right ) . Bellow : graph showing percentages of axes with : separation of AEs ( grey ) closely associated AEs ( grey ) . 414 axes analysed , from 11 nuclei . Bars , 10 μm in spreads 1 μm on insets .

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

B) Representative nuclear spread of zygotene-like Stag3 mutant spermatocytes. Nuclear spreads were immunostained for SYCP3 and ACA. Magnified views are indicated by dashed areas. Schematic representation on magnified univalents represents: separation of sister-AEs (centre) and close association of sister-AEs (right). Bellow: graph showing the percentages of axes with: separation of AEs (dark grey area) and closely associated AEs (light grey area). 414 axes analysed, from 11 nuclei. Bars, 10 μm in spreads and 1 μm on insets.


C ) Magnified views of zygotene - like Stag3 mutant univalents with LSAEs . spreads were immunostained for SYCP3 ACA . Schematic representation indicates sites of LSAEs . Bellow : graph showing percentages of axes with : LSAEs ( red ) , extensive separation of AEs ( grey ) closely associated AEs ( grey ) . 414 axes analysed , from 11 nuclei . Bars , 1 μm .

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

C) Magnified views of zygotene-like Stag3 mutant univalents with LSAEs. Nuclear spreads were immunostained for SYCP3 and ACA. Schematic representation indicates sites of LSAEs. Bellow: graph showing the percentages of axes with: LSAEs (red area), extensive separation of AEs (dark grey area) and closely associated AEs (light grey area). 414 axes analysed, from 11 nuclei. Bars, 1 μm.


D ) Representative spread of zygotene - like Smc1β − / − spermatocytes . spreads were immunostained for SYCP3 ACA . Magnified views are indicated by dashed areas . Schematic representation on magnified univalents represents sites of LSAEs . Bellow : graph showing percentages of axes with : LSAEs ( red ) , extensive separation of AEs ( grey ) closely associated AEs ( grey ) . 404 axes analysed , from 10 nuclei . Bars , 10 μm in spreads 1 μm on insets .

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

D) Representative nuclear spread of zygotene-like Smc1β−/− spermatocytes. Nuclear spreads were immunostained for SYCP3 and ACA. Magnified views are indicated by dashed areas. Schematic representation on magnified univalents represents sites of LSAEs. Bellow: graph showing the percentages of axes with: LSAEs (red area), extensive separation of AEs (dark grey area) and closely associated AEs (light grey area). 404 axes analysed, from 10 nuclei. Bars, 10 μm in spreads and 1 μm on insets.


E ) STED of representative zygotene - like Stag3 mutant Smc1β − / − univalents displaying LSAEs . spreads were immunostained for SYCP3 . Bars , 1μm .

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

E) STED images of representative zygotene-like Stag3 mutant and Smc1β−/− univalents displaying LSAEs. Nuclear spreads were immunostained for SYCP3. Bars, 1μm.


F ) Graph indicating inter - axis distances measured in wild - type pachytene bivalents ; pachytene - like Rec8 − / − zygotene - like Stag3 mutant Smc1β − / − univalents . Each measurement in wild - type Rec8 − / − corresponds to median of 3 distances measured along one homolog ( n = 60 ) . Each measurement in Stag3 mutant Smc1β − / − mutant axes correspond to 1 measured at sites of local separation of AEs ( n = 40 n = 20 , respectively ) . Horizontal lines indicate median .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graph indicating the inter-axis distances measured in wild-type pachytene bivalents; pachytene-like Rec8−/− and zygotene-like Stag3 mutant and Smc1β−/− univalents. Each measurement in wild-type and Rec8−/− corresponds to the median of 3 distances measured along one homolog (n=60). Each measurement in Stag3 mutant and Smc1β−/− mutant axes correspond to 1 distance measured at sites of local separation of AEs (n=40 and n=20, respectively). Horizontal lines indicate median.


G ) Graph indicating inter - axis distances measured at terminally centrally located sites of LSAEs in zygotene - like Stag3 mutant univalents . Each measurement corresponds to 1 measured at terminally ( n = 20 ) centrally ( n = 20 ) located sites of local separation of axial elements . Horizontal lines indicate median .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graph indicating the inter-axis distances measured at terminally and centrally located sites of LSAEs in zygotene-like Stag3 mutant univalents. Each measurement corresponds to 1 distance measured at terminally (n=20) and centrally (n=20) located sites of local separation of axial elements. Horizontal lines indicate median.


A ) Representative wild - type pachytene bivalents ; pachytene - like Rec8 − / − , zygotene - like Stag3 mutant Smc1β − / − univalents . spreads were immunostained for SYCP1 , SYCP3 ACA . Filled arrowheads indicate sites of LSAEs with detectable SYCP1 . Bars , 1 μm . Bellow : graphs showing percentages of sites of LSAEs with detectable SC assembly along sister - AEs of Stag3 mutant Smc1β − / − univalents ( n = 120 n = 68 , 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 O O O O O O O O O O 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 wild-type pachytene bivalents; pachytene-like Rec8−/−, zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP1, SYCP3 and ACA. Filled arrowheads indicate sites of LSAEs with detectable SYCP1. Bars, 1 μm. Bellow: graphs showing the percentages of sites of LSAEs with detectable SC assembly along sister-AEs of Stag3 mutant and Smc1β−/− univalents (n=120 and n=68, respectively).


B ) Representative zygotene - like Stag3 mutant univalents . spreads were immunostained for SYCP3 , ACA : SYCE1 , SYCE2 TEX12 . Filled arrowheads indicate SC assembly between sites of LSAEs . Bars , 1

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

B) Representative zygotene-like Stag3 mutant univalents. Nuclear spreads were immunostained for SYCP3, ACA and: SYCE1, SYCE2 and TEX12. Filled arrowheads indicate SC assembly between sites of LSAEs. Bars, 1 μm


C ) of signal distribution within sites of LSAEs , in Stag3 mutant spermatocytes . Signal distribution measured for SYCP3 : SYCP1 , SYCE1 , SYCE2 TEX12 . Empty arrowheads indicate signal peaks that correspond to each of two sister - chromatid axes , filled arrowheads indicate SYCP1 peaks .

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

C) Quantification of signal distribution within sites of LSAEs, in Stag3 mutant spermatocytes. Signal distribution measured for SYCP3 and: SYCP1, SYCE1, SYCE2 and TEX12. Empty arrowheads indicate signal intensity peaks that correspond to each of the two sister-chromatid axes, filled arrowheads indicate SYCP1 peaks.


A ) Representative zygotene - like Stag3 mutant Smc1β − / − univalents . spreads were immunostained for SYCP3 , SYCE1 REC8 . Bellow : graph showing percentages of Stag3 mutant axes with sites of LSAEs with without flanking REC8 foci . 78 axes with LSAEs were analysed . Bars , 1 μm .

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

A) Representative zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and REC8. Bellow: graph showing the percentages of Stag3 mutant axes with sites of LSAEs with and without flanking REC8 foci. 78 axes with LSAEs were analysed. Bars, 1 μm.


B ) Representative pachytene - like Rec8 − / − , zygotene - like Stag3 mutant Smc1β − / − univalents . spreads were immunostained for SYCP3 , SYCE1 RAD21L . Bars , 1 μm .

B) Representative pachytene-like Rec8−/−, zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and RAD21L. Bars, 1 μm.


C ) Representative zygotene - like Stag3 mutant Smc1β − / − univalents . spreads were immunostained for SYCP3 , SYCE1 RAD21 . Bars , 1 μm .

C) Representative zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and RAD21. Bars, 1 μm.


A ) Representative wild - type pachytene sex chromosomes . spreads of pachytene spermatocytes were immunostained for SYCP3 . Filled arrowheads indicate PAR . Dashed indicated in centre panel corresponds to magnified view on right panel . Bars , 10 μm in spreads 1 μm on insets .

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A) Representative wild-type pachytene sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3. Filled arrowheads indicate the PAR. Dashed area indicated in centre panel corresponds to the magnified view on the right panel. Bars, 10 μm in spreads and 1 μm on insets.


B ) Graph indicating inter - axis distances measured at sites of local separation of axial elements in zygotene - like Stag3 mutant univalents along pachytene wild - type X - chromosomes . Each measurement in X - chromosomes corresponds to median of 3 distances ( according to scheme in figure ) ( n = 54 ) . Each measurement in Stag3 mutant corresponds to 1 measured at sites with local separation of axial elements ( n = 40 ) . Horizontal lines indicate median . p < 0 . 0001 , obtained with two - Mann - Whitney . Bellow : schematic representation of XY pair : sister - AEs are coloured in black grey for easier visualization . Filled arrowhead indicates PAR . Purple bars in scheme indicate regions where inter - axis distances are measured .

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B) Graph indicating the inter-axis distances measured at sites of local separation of axial elements in zygotene-like Stag3 mutant univalents and along pachytene wild-type X-chromosomes. Each measurement in the X-chromosomes corresponds to the median of 3 distances (according to the scheme in figure) (n=54). Each measurement in Stag3 mutant corresponds to 1 distance measured at sites with local separation of axial elements (n=40). Horizontal lines indicate median. p < 0.0001, obtained with two-tailed Mann-Whitney test. Bellow: schematic representation of the XY pair: sister-AEs are coloured in black and grey for easier visualization. Filled arrowhead indicates PAR. Purple bars in scheme indicate regions where inter-axis distances are measured.


C ) Representative pachytene wild - type sex chromosomes . spreads of pachytene spermatocytes were immunostained for SYCP3 SYCE2 . Dashed indicated in centre panel corresponds to magnified view on right panel . Empty arrowhead indicates two discernable sister - AEs without SYCE2 . Bars , 10 μm in spreads 1 μm on insets .

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C) Representative pachytene wild-type sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3 and SYCE2. Dashed area indicated in centre panel corresponds to the magnified view on the right panel. Empty arrowhead indicates two discernable sister-AEs without SYCE2. Bars, 10 μm in spreads and 1 μm on insets.


A ) of REC8 distribution in representative pachytene wild - type sex chromosomes . spreads of pachytene spermatocytes were immunostained for SYCP3 REC8 . Bars , 10 μm in spreads 1 μm on insets . Magnified XY pair is indicated by dashed . Scatterplot showing number of REC8 foci per X - chromosome . 225 foci identified in 14 X - chromosomes . Horizontal indicates mean , error bars indicate s . d . 3 . 02 .

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A) Analysis of REC8 distribution in representative pachytene wild-type sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3 and REC8. Bars, 10 μm in spreads and 1 μm on insets. Magnified XY pair is indicated by dashed area. Scatterplot showing the number of REC8 foci per X-chromosome. 225 foci identified in 14 X-chromosomes. Horizontal line indicates the mean, error bars indicate s.d. 3.02.


B ) Top : representative computationally straightened pachytene wild - type X - chromosomes . spreads were immunostained for SYCP3 REC8 . Chromosomes were aligned at PAR , subdivided into 25 regular intervals . Bar , 1 μm . Bellow : histogram showing distribution of percentages of REC8 foci ( n = 225 ) among 25 regular intervals along 14 X - chromosomes . Dotted indicates mean .

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B) Top: representative computationally straightened pachytene wild-type X-chromosomes. Nuclear spreads were immunostained for SYCP3 and REC8. Chromosomes were aligned at the PAR, and subdivided into 25 regular intervals. Bar, 1 μm. Bellow: histogram showing the distribution of the percentages of REC8 foci (n=225) among 25 regular intervals along 14 X-chromosomes. Dotted line indicates mean.


C ) Histogram showing relative frequency of inter - REC8 distances along X - chromosomes . 211 inter - REC8 distances from 14 X - chromosomes . Grey represents 15 % of chromosome axis . respective cumulative distribution function is shown .

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C) Histogram showing the relative frequency of inter-REC8 distances along X-chromosomes.211 inter-REC8 distances from 14 X-chromosomes. Grey area represents 15% of the chromosome axis length. The respective cumulative distribution function is shown.


D ) Histogram showing best fit of observed inter - REC8 distances to gamma distribution . Green curve indicates best fit of observed inter - REC8 distances .  indicates value for which best fit was obtained , with estimated standard error 0 . 27 .

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D) Histogram showing the best fit of the observed inter-REC8 distances to the gamma distribution. Green curve indicates the best fit of the observed inter-REC8 distances.  indicates the value for which the best fit was obtained, with estimated standard error 0.27.


E ) Graph comparing median of REC8 foci with computationally predicted even random distributions . Median distances between foci subdivided in three categories ( 11 - 14 , 15 - 18 19 - 22 REC8 foci per chromosome ) . Purple asterisk indicates median of REC8 foci .

E) Graph comparing the median distance of REC8 foci with computationally predicted even and random distributions. Median distances between foci subdivided in three categories (11-14, 15-18 and 19-22 REC8 foci per chromosome). Purple asterisk indicates the median distance of all REC8 foci.


F ) Scatterplot comparing inter - REC8 distances along on X - chromosomes with distances between flanking REC8 foci at sites of LSAEs in Stag3 mutant . 211 inter - REC8 distances measured along X - chromosomes 10 distances measured between flanking REC8 foci in Stag3 mutant . Grey represents 15 % of chromosome axis . Horizontal lines indicate median . p < 0 . 0001 , obtained with two - Mann - Whitney .

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F) Scatterplot comparing inter-REC8 distances along on X-chromosomes with the distances between flanking REC8 foci at sites of LSAEs in the Stag3 mutant. 211 inter-REC8 distances measured along X-chromosomes and 10 distances measured between flanking REC8 foci in Stag3 mutant. Grey area represents 15% of the chromosome axis length. Horizontal lines indicate the median. p < 0.0001, obtained with two-tailed Mann-Whitney test.


A ) Representative yet - unsynapsed autosome from wild - type zygotene spermatocytes . spreads were immunostained for SYCP3 SYCE2 . Magnified autosome is indicated by dashed . Filled arrowheads indicate yet - unsynapsed axes regions . Bars , 10 μm in spreads 1 μm on

A) Representative yet-unsynapsed autosome from wild-type zygotene spermatocytes. Nuclear spreads were immunostained for SYCP3 and SYCE2. Magnified autosome is indicated by dashed area. Filled arrowheads indicate yet-unsynapsed axes regions. Bars, 10 μm in spreads and 1 μm on insets


B ) Representative unsynapsed autosome from wild - type zygotene spermatocytes . spreads were immunostained for SYCP3 , REC8 ACA . Magnified autosome is indicated by dashed . Bars , 10 μm in spreads 1 μm on insets .

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B) Representative unsynapsed autosome from wild-type zygotene spermatocytes. Nuclear spreads were immunostained for SYCP3, REC8 and ACA. Magnified autosome is indicated by dashed area. Bars, 10 μm in spreads and 1 μm on insets.


C ) Scatterplot comparing average number of REC8 foci along zygotene autosomes X - chromosomes . Number of REC8 foci were quantified along 15 zygotene autosomes 14 X - chromosomes . Each dot represents number of REC8 foci on each chromosome . Total number of foci identified : zygotene autosomes ( 343 ) , X - chromosomes ( 225 ) . Horizontal lines indicate mean , error bars indicate s . d . : 7 . 42 3 . 02 for zygotene autosomes X - chromosomes , respectively . p = 0 . 0060 , obtained with two - Mann - Whitney .

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C) Scatterplot comparing the average number of REC8 foci along zygotene autosomes and X-chromosomes. Number of REC8 foci were quantified along 15 zygotene autosomes and 14 X-chromosomes. Each dot represents the number of REC8 foci on each chromosome. Total number of foci identified: zygotene autosomes (343), X-chromosomes (225). Horizontal lines indicate the mean, error bars indicate s.d.: 7.42 and 3.02 for zygotene autosomes and X-chromosomes, respectively. p = 0.0060, obtained with two-tailed Mann-Whitney test.


D ) Histogram showing relative frequency of inter - REC8 distances along yet - unsynapsed zygotene autosomes . 294 inter - REC8 distances analyzed in 14 chromosomes . Grey represents 15 % of chromosome axis . respective cumulative distribution function is shown .

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D) Histogram showing the relative frequency of inter-REC8 distances along yet-unsynapsed zygotene autosomes. 294 inter-REC8 distances analyzed in 14 chromosomes. Grey area represents 15% of the chromosome axis length. The respective cumulative distribution function is shown.


E ) Scatterplot comparing measured inter - REC8 distances on yet - unsynapsed zygotene autosomes , X - chromosomes sites of LSAEs in Stag3 mutant . Number of inter - REC8 distances for yet - unsynapsed zygotene autosomes , X - chromosomes sites of local separation of axial elements in Stag3 mutant , n = 294 , n = 211 n = 10 , respectively . Horizontal lines indicate median . p < 0 . 0001 , obtained with two - Mann - Whitney .

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E) Scatterplot comparing the measured inter-REC8 distances on yet-unsynapsed zygotene autosomes, X-chromosomes and sites of LSAEs in Stag3 mutant. Number of inter-REC8 distances for yet-unsynapsed zygotene autosomes, X-chromosomes and sites of local separation of axial elements in Stag3 mutant, n=294, n=211 and n=10, respectively. Horizontal lines indicate the median. p < 0.0001, obtained with two-tailed Mann-Whitney test.


Immunoblot of whole lysates from multiple human lineage - committed - lines using a C - terminal anti - JARID2 antibody . of canonical form is 140kDa but an additional strong band was detected at around ~ 80kDa . blot is divided according to - types , which are indicated in figure .

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Immunoblot of whole cell lysates from multiple human lineage-committed cell-lines using a C-terminal anti-JARID2 antibody. The size of canonical form is 140kDa but an additional strong band was detected at around ~80kDa. The blot is divided according to cell-types, which are indicated in the figure.


qPCR measurements of JARID2 mRNA in different lines . were measured relative to 18S rRNA . Means SEs are calculated over at least 2 independent experiments 3 technical replicates . data is represented as mean ± SE .

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qPCR measurements of JARID2 mRNA levels in different cell lines. The RNA levels were measured relative to 18S rRNA. Means and SEs are calculated over at least 2 independent experiments and 3 technical replicates. The data is represented as mean ± SE.


Immunoblot showing that ~ 80kDa band disappears upon treatment of HEK293T HaCaT with siRNAs against exon 3 exon 15 of JARID2 . Non - silencing control ( NSC ) was used as a transfection control .

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Immunoblot showing that the ~80kDa band disappears upon treatment of HEK293T and HaCaT cells with siRNAs against exon 3 and exon 15 of JARID2 RNA. Non-silencing control (NSC) was used as a transfection control.


qPCR measurements of ( n = 3 ) for three isoforms of JARID2 in HaCaT . are plotted relative to 18S rRNA . Level of JARID2 isoform - 1 is significantly higher than that of isoform - 2 ( * * * P < 0 . 001 ) isoform - 3 ( * * * * P < 0 . 0001 ) .

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qPCR measurements of RNA levels (n=3) for the three isoforms of JARID2 in HaCaT cells. The RNA levels are plotted relative to 18S rRNA. Level of JARID2 isoform-1 is significantly higher than that of isoform-2 (***P<0.001) and isoform-3 (****P<0.0001).


Agarose gel showing RT - PCR products amplified using primers in exon3 exon15 ( 2 . 9kb ) as well as RT - PCR product corresponding to JARID2 isoform - 1 ( 3 . 7kb ) amplified using first ( exon 1 ) last exon ( exon18 ) . Only one product at right is observed indicating that it is unlikely that JARID2 might be a product of an alternatively spliced isoform of JARID2 .

Agarose gel showing RT-PCR products amplified using primers in exon3 and exon15 (2.9kb) as well as RT-PCR product corresponding to JARID2 isoform-1 (3.7kb) amplified using the first (exon 1) and the last exon (exon18). Only one product at the right size is observed indicating that it is unlikely that JARID2 might be a product of an alternatively spliced isoform of JARID2.


CRISPR - Cas9 knockout of JARID2 using a sgRNA guide designed to target main translation start site as seen in ( E ) . Immunoblot revealed that ~ 80kDa form was removed from JARID2 KO lines .

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CRISPR-Cas9 knockout of JARID2 using a sgRNA guide designed to target main translation start site as seen in (E). Immunoblot revealed that ~80kDa form was removed from JARID2 KO lines.


Immunoblot after a full JARID2 isoform - 1 was expressed from an exogenous vector in HaCaT showed an increase ( 2 - 3 fold ) in ~ 80kDa level .

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Immunoblot after a full length JARID2 isoform-1 was expressed from an exogenous vector in HaCaT cells showed an increase (2-3 fold) in ~80kDa level.


Immunoprecipitation using anti - JARID2 antibody followed by mass spectrometry identification of ~ 80kDa band detected JARID2 peptides spanning only C - terminus of JARID2 . identified peptides are shown as red arrows on schematic of JARID2 .

Immunoprecipitation using anti-JARID2 antibody and followed by mass spectrometry identification of the ~80kDa band detected JARID2 peptides spanning only C-terminus of JARID2. The identified peptides are shown as red arrows on schematic of JARID2 sequence.


Immunoblot showing increase in expression of differentiation markers Involucrin ( IVL ) Transglutaminase - 1 ( TGase - 1 ) as differentiation progressed indicating that keratinocyte differentiation protocol was successful . on day 0 ( D0 ) , day 1 ( D1 ) , day 3 ( D3 ) day 6 ( D6 ) of differentiation are shown . Immunoblot for JARID2 during D0 , D1 , D3 D6 of differentiation ( as in B ) are shown .

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Immunoblot showing increase in expression levels of differentiation markers Involucrin (IVL) and Transglutaminase-1 (TGase-1) as differentiation progressed indicating that keratinocyte differentiation protocol was successful. Protein levels on day 0 (D0), day 1 (D1), day 3 (D3) and day 6 (D6) of differentiation are shown. Immunoblot for JARID2 during D0, D1, D3 and D6 of differentiation (as in B) are shown.


Effect of JARID2 removal on of differentiation markers Involucrin ( IVL ) , Keratin - 1 ( KRT1 ) Keratin - 10 ( KRT10 ) mRNAs as measured in qPCR experiment relative to 18S rRNA rescue using exogenous expression of ΔN - JARID2 . effect of exogenous expression of full - JARID2 ( FL - JARID2 ) in JARID2 knockout is also shown . Data from wildtype HaCaTs , two independent JARID2 knockout ( KO1 KO2 ) HaCaT lines as well as KO exogenously expressing empty vector control , ΔN - JARID2 FL - JARID2 are shown . of Involucrin ( IVL ) , Keratin - 1 ( KRT1 ) Keratin - 10 ( KRT10 ) mRNAs as above but measured on day 3 of differentiation .

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Effect of JARID2 removal on levels of differentiation markers Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as measured in qPCR experiment relative to 18S rRNA and the rescue using exogenous expression of ΔN-JARID2. The effect of exogenous expression of full-length JARID2 (FL-JARID2) in JARID2 knockout is also shown. Data from wildtype HaCaTs, two independent JARID2 knockout (KO1 and KO2) HaCaT lines as well as KO cells exogenously expressing empty vector control, ΔN-JARID2 and FL-JARID2 are shown. Levels of Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as above but measured on day 3 of differentiation.


A metagene plot showing enrichment of full - JARID2 in human induced pluripotent at down - regulated genes ( blue ) as compared to up - regulated genes ( grey ) in JARID2 KO . plot is centered on Transcription Start Sites ( TSS ) of genes from TSS is indicated on x - axis .

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A metagene plot showing enrichment of full-length JARID2 in human induced pluripotent cells at down-regulated genes (blue) as compared to up-regulated genes (grey) in JARID2 KO cells. The plot is centered on Transcription Start Sites (TSS) of genes and distance from TSS is indicated on the x-axis.


Metagene plots of average enrichment of EZH2 in ES keratinocytes at down - regulated genes ( blue ) as compared to up - regulated genes ( grey ) in JARID2 KO . plots are centered on Transcription Start Sites ( TSS ) of genes from TSS is indicated on x - axes .

Metagene plots of average enrichment of EZH2 in ES cells and keratinocytes at down-regulated genes (blue) as compared to up-regulated genes (grey) in JARID2 KO cells. The plots are centered on Transcription Start Sites (TSS) of genes and distance from TSS is indicated on the x-axes.


of H3K27me3 in WT , JARID2 KO lines JARID2 KO lines expressing ΔN - JARID2 . Histone H3 Ponceau is used as a loading control .

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Levels of H3K27me3 in WT, JARID2 KO lines and JARID2 KO lines expressing ΔN-JARID2. Histone H3 and Ponceau staining is used as a loading control.


A bar - plot showing of down - up - regulated genes among genes which lose H3K27me3 vs which do not lose H3K27me3 modification during differentiation .

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A bar-plot showing percentage of down- and up-regulated genes among genes which lose H3K27me3 vs which do not lose H3K27me3 modification during differentiation.


Geometric mean - centered , hierarchical cluster heat map from microarray data . 1077 annotated lncRNAs ( p < 0 . 05 ) were represented in liver CSC ( CD13 + CD133 + ) compared with non - CSC ( CD13 - CD133 - ) sorted from three HCC of three . Top ten upregulated lncRNAs in CSCs are shown .

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Geometric mean-centered, hierarchical cluster heat map from microarray data. 1077 annotated lncRNAs (p<0.05) were represented in liver CSC (CD13+CD133+) compared with non-CSC (CD13-CD133-) cells sorted from three HCC primary cells of three patients. Top ten upregulated lncRNAs in CSCs are shown.


In situ hybridization of HAND2 - AS1 in HCC tissues . HAND2 - AS1 is shown in tissues ( left panel ) . Quantitation of HAND2 - AS1 + in liver tissues was examined in 10 high - power fields ( HPF ) of sections from ten different HCC ( right panel ) . Results are shown as means ± . Scale bar , 100 μm .

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In situ hybridization of HAND2-AS1 in HCC tumor tissues. HAND2-AS1 staining is shown in tumor tissues (left panel). Quantitation of HAND2-AS1+ cells in liver tissues was examined in 10 high-power fields (HPF) of sections from ten different HCC patients (right panel). Results are shown as means ± SEM. Scale bar, 100 μm.


Macroscopic appearance of livers in 12 - month - old DEN - treated lncHand2 + / + lncHand2 knockout ( lncHand2 - / - ) mice ( left panel ) . Black arrows indicate liver tumors . Quantitation of foci numbers areas in DEN - treated livers ( right panel ) . Results are shown as means ± SD ( n = 12 ) .

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Macroscopic appearance of livers in 12-month-old DEN-treated lncHand2+/+ and lncHand2 knockout (lncHand2-/-) mice (left panel). Black arrows indicate liver tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers (right panel). Results are shown as means ± SD (n=12).


Representative H & E immunohistological with Ki67 antibody of liver sections of 12 - month - old DEN - treated lncHand2 + / + lncHand2 - / - mice . Scale bar , 100 μm .

Representative H&E and immunohistological staining with Ki67 antibody of liver sections of 12-month-old DEN-treated lncHand2+/+ and lncHand2-/- mice. Scale bar, 100 μm.


Quantitation of Ki67 - positive in non - treated DEN - treated mice . Bars represent average percentages SD of positively for Ki67 examined in 10 HPF of sections from five different mice . Data are shown as means ± SD .

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Quantitation of Ki67-positive cells in non-treated and DEN-treated mice. Bars represent average percentages and SD of cells positively staining for Ki67 cells examined in 10 HPF of sections from five different mice. Data are shown as means ± SD.


Representative immunofluorescence of livers in 5 . 5 - month - old DEN - treated untreated lncHand2RFP reporter mice for indicated molecules ( lower panel ) . Scale bar , 100 μm . Upper panel : scheme of targeting strategy for IRES - RFP knockin allele .

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Representative immunofluorescence staining of livers in 5.5-month-old DEN-treated and untreated lncHand2RFP reporter mice for indicated molecules (lower panel). Scale bar, 100 μm. Upper panel: scheme of targeting strategy for IRES-RFP knockin allele.


HAND2 - AS1 knockout causes a declined oncosphere - forming capacity in HCC . right panel represents statistical results as means ± SD ( n = 3 per group ) . Overexpression of HAND2 - AS1 ( oelnc ) rescued sphere formation reduced by HAND2 - AS1 deletion . Scale bar , 100 μm .

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HAND2-AS1 knockout causes a declined oncosphere-forming capacity in HCC cells. The right panel represents statistical results as means ± SD (n=3 per group). Overexpression of HAND2-AS1 (oelnc) rescued the sphere formation reduced by HAND2-AS1 deletion. Scale bar, 100 μm.


CD13 + CD133 + ( CSC ) subpopulations were detected in HAND2 - AS1 knockout by FACS . Results are shown as means ± SD ( n = 4 per group ) .

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CD13+CD133+ (CSC) subpopulations were detected in HAND2-AS1 knockout cells by FACS analysis. Results are shown as means ± SD (n=4 per group).


Limiting diluted HAND2 - AS1 depletion or Ctrl HCC were subcutaneously implanted into BALB / c nude mice . n = 12 for each group .

Limiting diluted HAND2-AS1 depletion or Ctrl HCC cells were subcutaneously implanted into BALB/c nude mice. n=12 for each group.


Estimated frequency of TICs in HAND2 - AS1 deficiency control after first transplantation using extreme limiting dilution . Data are shown as mean 95 % confidence interval . ( n = 12 grafted tumors per dilution ) .

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Estimated frequency of TICs in HAND2-AS1 deficiency and control cells after the first transplantation using the extreme limiting dilution analysis. Data are shown as the mean and 95% confidence interval. (n=12 grafted tumors per dilution).


Estimated frequency of TICs in HAND2 - AS1 deficiency control HCC during serial transplantations . Grey indicates 95 % CI .

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Estimated frequency of TICs in HAND2-AS1 deficiency and control HCC cells during serial transplantations. Grey area indicates the 95% CI.


Estimated frequency of TICs in different populations after first transplantation ( n = 15 grafted tumors per dilution ) . Data represent mean 95 % confidence interval .

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Estimated frequency of TICs in different populations after the first transplantation (n=15 grafted tumors per dilution). Data represent the mean and 95% confidence interval.


Representative whole - imaging of Huh7 - Luc transduced with control or HAND2 - AS1 KO # 1 or KO # 2 vectors . of numbers of 6 after orthotopically implanted to B - NSG mice . Data are shown as means ± SD . One - way ANOVA with Dunnett ' s correction for multiple comparisons .

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Representative whole-body imaging of Huh7-Luc cells transduced with control or HAND2-AS1 KO #1 or KO #2 vectors. Quantification of tumor numbers of 6 weeks after tumor cells orthotopically implanted to B-NSG mice. Data are shown as means ± SD. One-way ANOVA with Dunnett's correction for multiple comparisons.


HAND2 - AS1 expression ( normalized to 18S rRNA ) in livers from BALB / c nude mice subcutaneously transplanted xenograft HCC then intraperitoneally administered 25 mg kg − 1 ASOs on days 15 , 20 , 25 , 30 35 ( n = 5 per group ) . Three ASOs ( 1 , 2 , 3 ) against HAND2 - AS1 were used to treat mice showed similar depletion effects . Results are shown as means ± SD . n = 6 mice per group .

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HAND2-AS1 expression levels (normalized to 18S rRNA) in livers from BALB/c nude mice subcutaneously transplanted xenograft primary HCC cells then intraperitoneally administered 25 mg kg−1 ASOs on days 15, 20, 25, 30 and 35 (n = 5 per group). Three ASOs (1, 2, 3) against HAND2-AS1 were used to treat mice and showed similar depletion effects. Results are shown as means ± SD. n=6 mice per group.


Mice were killed at 40th day after HCC injection , tumors were excised weighed . Representative tumors are shown . Scale bar , 1 cm . Statistical data are shown as means ± SD ( n = 6 mice per group ) ( right panel ) .

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Mice were killed at the 40th day after HCC cell injection, and the tumors were excised and weighed. Representative tumors are shown. Scale bar, 1 cm. Statistical data are shown as means ± SD (n = 6 mice per group) (right panel).


HAND2 - AS1 overexpression enhances capacity of oncosphere formation . Scale bar , 100 μm . oe : overexpression . Right panel : Data are shown as means ± SD ( n = 3 per group ) .

HAND2-AS1 overexpression enhances the capacity of oncosphere formation. Scale bar, 100 μm. oe: overexpression. Right panel: Data are shown as means ± SD (n = 3 per group).


Estimated frequency of TICs in HAND2 - AS1 overexpression control HCC during serial transplantations .

Estimated frequency of TICs in HAND2-AS1 overexpression and control HCC cells during serial transplantations.


Representative whole - imaging of Huh7 - Luc transduced with control or HAND2 - AS1 overexpression vectors . for numbers one month after orthotopically implanted to B - NSG mice . ( n = 5 mice per group ) .

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Representative whole-body imaging of Huh7-Luc cells transduced with control or HAND2-AS1 overexpression vectors. Quantification for tumor numbers one month after tumor cells orthotopically implanted to B-NSG mice. (n = 5 mice per group).


Biotin - pulldowns were performed with extracts of oncosphere using full - HAND2 - AS1 transcript ( sense ) , antisense , one HAND2 - AS1 intron control , followed by mass spectrometry . Band 1 : INO80 , band 2 : RUVBL2 .

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Biotin-RNA pulldowns were performed with nuclear extracts of oncosphere cells using full-length HAND2-AS1 transcript (sense), antisense, and one HAND2-AS1 intron control, followed by mass spectrometry. Band 1: INO80, band 2: RUVBL2.


Two components of INO80 were confirmed by immunoblotting .

Two core components of the INO80 complex were confirmed by immunoblotting.


Immunoprecipitation assays of biotin - labeled CHIRP probes incubated with liver CSC lysates .

Immunoprecipitation assays of biotin-labeled CHIRP probes incubated with liver CSC lysates.


HAND2 - AS1 was visualized by FISH , followed by immunofluorescence of INO80 in HCC oncosphere . Scale bar , 100 μm .

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HAND2-AS1 was visualized by RNA FISH, followed by immunofluorescence staining of INO80 in HCC primary oncosphere cells. Scale bar, 100 μm.


Mapping of INO80 - binding domains of HAND2 - AS1 . Schematic diagram of HAND2 - AS1 full - truncated fragments ( top panel ) ; Western blot of INO80 RUVBL2 in pulldown samples by different HAND2 - AS1 fragments ( middle panel ) ; Different HAND2 - AS1 fragments ( bottom panel ) .

Mapping analysis of INO80-binding domains of HAND2-AS1. Schematic diagram of HAND2-AS1 full-length and truncated fragments (top panel); Western blot of INO80 and RUVBL2 in RNA pulldown samples by different HAND2-AS1 fragments (middle panel); Different HAND2-AS1 fragments (bottom panel).


EMSA of biotin - labeled HAND2 - AS1 ( nt 83 - 242 ) probes incubated with INO80 .

EMSA of biotin-labeled HAND2-AS1 (nt 83-242) probes incubated with INO80 protein.


Binding affinity of HAND2 - AS1 with INO80 was determined by five independent EMSA assays . Non - linear regression curves were generated by GraphPad Prism . Results are shown as means ± SD .

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Binding affinity of HAND2-AS1 with INO80 was determined by five independent EMSA assays. Non-linear regression curves were generated by GraphPad Prism. Results are shown as means ± SD.


Interaction regions of INO80 with HAND2 - AS1 mutations at nt 83 - 242 of exon 2 were detected using pulldown assays . LR , loop region .

Interaction regions of INO80 with HAND2-AS1 mutations at nt 83-242 of exon 2 were detected using RNA pulldown assays. LR, loop region.


Representative whole - imaging of Huh7 - Luc transduced with control or INO80 shRNA # 1 or shRNA # 2 vectors . of numbers of 4 after orthotopically implanted to B - NSG mice . Data are shown as means ± SD ( n = 5 mice per group ) . One - way ANOVA with Dunnett ' s correction for multiple comparisons .

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Representative whole-body imaging of Huh7-Luc cells transduced with control or INO80 shRNA #1 or shRNA #2 vectors. Quantification of tumor numbers of 4 weeks after tumor cells orthotopically implanted to B-NSG mice. Data are shown as means ± SD (n = 5 mice per group). One-way ANOVA with Dunnett's correction for multiple comparisons.


Macroscopic appearance of livers in 10 . 5 - month - old DEN - treated Ino80flox / flox ( Ino80 + / + ) Ino80 knockout ( Ino80 - / - ) ( left panel ) . Black arrows indicate tumors . Quantitation of foci numbers areas in DEN - treated livers . Results are shown as means ± SD ( n = 10 ) .

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Macroscopic appearance of livers in 10.5-month-old DEN-treated Ino80flox/flox (Ino80+/+) and Ino80 knockout (Ino80-/-) (left panel). Black arrows indicate tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers. Results are shown as means ± SD (n=10).


results for HAND2 - AS1 or INO80 deficiency in HCC .

Heatmap results for HAND2-AS1 or INO80 deficiency in HCC cells.


on GSEA results , significantly changed genes due to depletion of HAND2 - AS1 INO80 were attributed to BMP signaling in HCC . NES , normalized enrichment score ; FWER , familywise error rate .

Based on GSEA results, significantly changed genes due to depletion of HAND2-AS1 and INO80 were attributed to the BMP signaling in HCC primary cells. NES, normalized enrichment score; FWER, familywise error rate.


ChIRP - seq of HAND2 - AS1 genomic binding at target sites in liver CSCs , using LacZ probes as negative control . A 10 - kb interval centered on called HAND2 - AS1 peak is shown .

ChIRP-seq analysis of HAND2-AS1 genomic binding at target sites in liver CSCs, using LacZ probes as negative control. A 10-kb interval centered on the called HAND2-AS1 peak is shown.


Representative ChIRP - seq of HAND2 - AS1 binding of BMPR1A .

Representative ChIRP-seq of HAND2-AS1 binding of BMPR1A.


Gel of HAND2 - AS1 binding BMPR1A promoter in liver CSCs . LacZ PVT1 served as negative controls .

Gel analysis of HAND2-AS1 RNA binding BMPR1A promoter in liver CSCs. LacZ and PVT1 served as negative controls.


HAND2 - AS1 deletion in HCC decreases BMPR1A expression inactivates BMP signaling .

HAND2-AS1 deletion in HCC cells decreases BMPR1A expression and inactivates BMP signaling.


ChIRP - immunoblotting of BMPR1A interaction with INO80 in control HAND2 - AS1 - knockout liver CSCs .

ChIRP-immunoblotting analysis of BMPR1A interaction with the INO80 complex in control and HAND2-AS1-knockout liver CSCs.


EMSA analysis of the interaction of HAND2-AS1, INO80 and RUVBL2 with the BMPR1A promoter region.


BMPR1A promoter binding region to HAND2 - AS1 / INO80 was deleted ( BMPR1A - p - KO ) in liver CSCs using lenti - Cas9 , followed by HAND2 - AS1 INO80 overexpression ( oeHAND2 - AS1 , oeINO80 ) for 36 h . BMPR1A mRNA were examined by realtime PCR . Data are shown as means ± SD . n = 5 . * * P < 0 . 01 by two - Student ' s t .

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BMPR1A promoter binding region to HAND2-AS1/INO80 was deleted (BMPR1A-p-KO) in liver CSCs using lenti-Cas9, followed by HAND2-AS1 and INO80 overexpression (oeHAND2-AS1, oeINO80) for 36 h. BMPR1A mRNA levels were examined by realtime PCR. Data are shown as means ± SD. n=5. ** P < 0.01 by two-tailed Student's t test.


BMP signaling was examined in INO80 - silenced liver CSCs by Western blot .

BMP signaling was examined in INO80-silenced liver CSCs by Western blot.


BMPR1A is highly expressed in HCC tissues provided by Wang ' s cohort ( GSE14520 ) .

BMPR1A is highly expressed in HCC tumor tissues provided by Wang's cohort (GSE14520).


Kaplan - Meier analyses of liver cancer outcomes in cancer genome atlas ( TCGA ) . P value for Kaplan - Meier curves was determined using a Mantel - Cox log - rank .

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Kaplan-Meier analyses of liver cancer outcomes in the cancer genome atlas (TCGA). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test.


Representative immunohistochemistry of BMPR1A was detected in HCC samples . Scale bar , 100 μm .

Representative immunohistochemistry staining of BMPR1A was detected in HCC samples. Scale bar, 100 μm.


BMPR1A overexpression rescues sphere formation ability reduced by HAND2 - AS1 depletion in HCC samples . Representative sphere formation is shown on left panel . Scale bar , 100 μm . Percentages of sphere - forming were calculated as means ± SD ( right panel ) . n = 4 .

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BMPR1A overexpression rescues sphere formation ability reduced by HAND2-AS1 depletion in HCC samples. Representative sphere formation is shown on the left panel. Scale bar, 100 μm. Percentages of sphere-forming cells were calculated as means ± SD (right panel). n=4.


BMPR1A depletion reduces - initiating capacity . n = 12 mice per group .

BMPR1A depletion reduces tumor-initiating capacity. n=12 mice per group.


Representative whole - imaging of Huh7 - Luc transduced with scramble ( Ctrl ) or BMPR1A shRNAs . for numbers of 7 after orthotopically implanted to B - NSG mice . Calculated data are shown as means ± SD ( n = 5 mice per group ) .

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Representative whole-body imaging of Huh7-Luc cells transduced with scramble (Ctrl) or BMPR1A shRNAs. Quantification for tumor numbers of 7 weeks after tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5 mice per group).


Macroscopic appearance of livers from 10 - month - old mice treated by DEN or vehicle . Scale bar , 1 cm . Black arrows indicates tumors .

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Macroscopic appearance of livers from 10-month-old mice treated by DEN or vehicle. Scale bar, 1 cm. Black arrows indicates tumors.


Representative H & E immunohistological with Ki67 antibody in liver sections of 10 - month - old DEN - treated WT Bmpr1a - / - mice . Scale bar , 100 μm . Right : Quantitation of Ki67 - positive in DEN - treated mice . Data are shown as means ± SD . n = 5 .

Representative H&E and immunohistological staining with Ki67 antibody in liver sections of 10-month-old DEN-treated WT and Bmpr1a-/- mice. Scale bar, 100 μm. Right: Quantitation of Ki67-positive cells in DEN-treated mice. Data are shown as means ± SD. n=5.


BMP signaling inhibitor noggin decreased sphere formation . Scale bar , 100 μm . Right : Data are shown as means ± SD ( n = 4 ) .

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BMP signaling inhibitor noggin decreased tumor sphere formation. Scale bar, 100 μm. Right: Data are shown as means ± SD (n=4).


Representative whole - imaging of Huh7 - Luc in different groups . Mice treated with scramble ASOs / or siRNAs obtained similar results to vehicle treatment . n ≥ 5 mice in each group .

Representative whole-body imaging of Huh7-Luc cells in different groups. Mice treated with scramble ASOs and/or siRNAs obtained similar results to vehicle treatment. n≥5 mice in each group.


of numbers treated with ASOs / or siRNAs after Huh7 - Luc orthotopically implanted to B - NSG mice . Calculated data are shown as means ± SD ( n = 5 ) .

Quantification of tumor numbers treated with ASOs and/or siRNAs after Huh7-Luc tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5).


Tumors were excised from livers of PDX liver cancer models . Scale bar , 0 . 5 cm .

Tumors were excised from livers of PDX liver cancer models. Scale bar, 0.5 cm.


Representative H & E in liver sections from - bearing mice treated with ASO1 siBMPR1A in orthotopic transplants of tumors from HCC # 50 HCC # 51 . Scale bar , 100 μm .

Representative H&E staining in liver sections from tumor-bearing mice treated with ASO1 and siBMPR1A in orthotopic transplants of tumors from HCC#50 and HCC#51. Scale bar, 100 μm.


Representative immunohistological with Ki67 antibody of - bearing mice treated with ASO1 siBMPR1A . Scale bar , 100 μm . Lower panel : Quantitation of Ki67 - positive in different groups . Data are shown as means ± SD ( n = 5 per group ) .

Representative immunohistological staining with Ki67 antibody of tumor-bearing mice treated with ASO1 and siBMPR1A. Scale bar, 100 μm. Lower panel: Quantitation of Ki67-positive cells in different groups. Data are shown as means ± SD (n=5 per group).


Northern blotting of HAND2 - AS1 immunobloting of BMPR1A in tumors from PDX HCC # 50 after treatment .

Northern blotting of HAND2-AS1 and immunobloting of BMPR1A in tumors from PDX HCC#50 after treatment.


Kaplan - Meier survival of B - NSG mice orthotopically transplanted with PDXs HCC # 50 ( I ) HCC # 51 ( J ) . Treatment with ASO1 combined siBMPR1A causes synergistic therapeutic effect than each treatment alone ( n = 10 per group ) . P value for Kaplan - Meier curves was determined using a Mantel - Cox log - rank .

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Kaplan-Meier survival analysis of B-NSG mice orthotopically transplanted with PDXs HCC#50 (I) and HCC#51 (J). Treatment with ASO1 combined siBMPR1A causes synergistic therapeutic effect than each treatment alone (n = 10 per group). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test.


Time lapse of T6SS activity in tagged parental ( VipA - mCherry2 ) ∆ tssA mutant background . parental forms multiple dynamic structures per . ∆ tssA mutant predominantly forms dynamic sheath spots ( arrow ) few WT - like sheath structures ( blue arrow ) . Scale bars : 2 µm .

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Time lapse images of T6SS activity in tagged parental strain (VipA-mCherry2) and ∆tssA mutant background. The parental strain forms multiple dynamic structures per cell. ∆tssA mutant predominantly forms dynamic sheath spots (white arrow) and few WT-like sheath structures (blue arrow). Scale bars: 2 µm.


H2 - T6SS dynamics in tagged parental ( ∆ retS TssB2 - mCherry2 , referred to as WT H2 ) ΔtssA2PA . Blue arrow indicates extending contracting T6SS sheath structures , arrow points to dynamic sheath spots . WT H2 harbors multiple dynamic sheath structures per , while ΔtssA2PA displays very few dynamic spots or extended contracting structures . Scale bars : 2 µm .

H2-T6SS dynamics in tagged parental strain (∆retS TssB2-mCherry2, referred to as WT H2) and ΔtssA2PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. The WT H2 strain harbors multiple dynamic sheath structures per cell, while the ΔtssA2PA strain displays very few dynamic spots or extended and contracting structures. Scale bars: 2 µm.


Time lapse of T6SS activity in tagged ( VipA - mCherry2 TssAVC - mNeonGreen ) . arrow indicates path of TssAVC localization during one cycle of T6SS dynamics . Scale bar : 1 µm .

Time lapse images of T6SS activity in tagged strain (VipA-mCherry2 TssAVC-mNeonGreen). White arrow indicates path of TssAVC localization during one cycle of T6SS dynamics. Scale bar: 1 µm.


Kymographs of dynamics observed for TssAVC . Frequency of specific dynamics is indicated in upper right corner of merge image . ( B ) Sheath TssAVC dynamics from A . TssAVC localizes to T6SS initiation site to distal end of growing sheath ( arrow ) . ( C ) Once sheath reaches periphery , TssAVC dissociates from distal end ( arrow ) sheath stays extended a prolonged period of time . ( D ) TssAVC dissociates from growing sheath structure re - associates at a later time point ( arrows ) to continue catalysis of polymerization . ( E ) TssAVC stays attached to sheath distal end after contraction ( arrow ) .

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Kymographs of dynamics observed for TssAVC. Frequency of specific dynamics is indicated in the upper right corner of the merge image. (B) Sheath and TssAVC dynamics from A. TssAVC localizes to T6SS initiation site and to distal end of growing sheath (white arrow). (C) Once sheath reaches cell periphery, TssAVC dissociates from distal end (white arrow) and sheath stays extended a prolonged period of time. (D) TssAVC dissociates from growing sheath structure and re-associates at a later time point (white arrows) to continue catalysis of polymerization. (E) TssAVC stays attached to sheath distal end after contraction (white arrow).


microscopy of T6SS dynamics in a double tagged ( ∆ retS TssB2 - mCherry2 TssA2PA - mNeonGreen ) . arrow indicates full cycle of extending contracting T6SS sheath . Scale bar : 1 µm .

Fluorescence microscopy of T6SS dynamics in a double tagged strain (∆retS TssB2-mCherry2 TssA2PA-mNeonGreen). White arrow indicates full cycle of extending and contracting T6SS sheath. Scale bar: 1 µm.


Kymographs of TssB2 TssA2PA dynamics . TssA2PA localizes at sheath initiation site on distal end during polymerization ( G ) , dissociates before contraction ( H ) , dissociates re - associated during sheath extension ( I ) or stays attached to distal end after contraction ( J ) .

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Kymographs of TssB2 and TssA2PA dynamics. TssA2PA localizes at sheath initiation site and on distal end during polymerization (G), dissociates before contraction (H), dissociates and re-associated during sheath extension (I) or stays attached to distal end after contraction (J).


Top view ( XY - plane ) side view ( XZ - plane ) of TssAVC Nt2 domain cryo - EM reconstruction ( pink , non transparent ) shown with fitted Nt2 - dimer model ( left ) . Partial Nt2 - CTD Nt1 - Nt2 linker densities are highlighted with black arrows . Top side views of ribbon diagram of Nt2 - dimer ( middle ) . Representative 2D class averages of TssAVC particles with visible connections between Nt2 - dimer CTD ( top right ) , representative 2D class averages of Nt2 - dimer particles , Nt2 - CTD linker Nt1 - Nt2 linker densities are highlighted with blue yellow arrows ( bottom right ) .

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Top view (XY-plane) and side view (XZ-plane) of the TssAVC Nt2 domain cryo-EM reconstruction (pink, non transparent) shown with fitted Nt2-dimer model (left). Partial Nt2-CTD and Nt1-Nt2 linker densities are highlighted with black arrows. Top and side views of ribbon diagram of Nt2-dimer (middle). Representative 2D class averages of TssAVC particles with visible connections between Nt2-dimer and CTD (top right), representative 2D class averages of Nt2-dimer particles, Nt2-CTD linker and Nt1-Nt2 linker densities are highlighted with blue and yellow arrows (bottom right).


∆ tagA mutant displays long bent but still dynamic structures . Scale bar : 2 µm . Frames from time lapse movies showing sheath dynamics in ∆ tagA mutant . Sheath polymerization does not stop when distal end reaches opposing ( arrow ) . Continued polymerization causes sheath to tilt distal end to move towards pole , ultimately effecting bending of whole structure . C . Sheath detaches from its anchor ( orange arrow ) , keeps extending to maximum then contracts . Scale bars : 1 µm .

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The ∆tagA mutant displays long and bent but still dynamic structures. Scale bar: 2 µm. Frames from time lapse movies showing sheath dynamics in the ∆tagA mutant. Sheath polymerization does not stop when the distal end reaches opposing membrane (white arrow). Continued polymerization causes the sheath to tilt and distal end to move towards the bacterial cell pole, ultimately effecting bending of the whole structure. C. Sheath detaches from its membrane anchor (orange arrow), keeps extending to maximum cell length and then contracts. Scale bars: 1 µm.


Time lapse series of T6SS activity in tagged ( VipA - mCherry2 TagAVC - mNeonGreen ) . Orange arrow indicates sheath distal end , arrow indicates initiation site of another T6SS sheath . Scale bar : 1 µm .

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Time lapse series of T6SS activity in tagged strain (VipA-mCherry2 TagAVC-mNeonGreen). Orange arrow indicates sheath distal end, white arrow indicates initiation site of another T6SS sheath. Scale bar: 1 µm.