( e ) Representative WB analysis of IGF2R and GAPDH expression in total protein lysates of the human ( healthy and DMD ) and murine ( C57Bl6 / J and mdx ) TA and VM muscles ( n = 2 per healthy and DMD ; n = 10 mice per group ) ; quantifications are expressed as the IGF2R / GAPDH ratio in arbitrary units .

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

(e) Representative WB analysis of IGF2R and GAPDH expression in total protein lysates of the human (healthy and DMD) and murine (C57Bl6/J and mdx) TA and VM muscles (n=2 per healthy and DMD; n=10 mice per group); quantifications are expressed as the IGF2R/GAPDH ratio in arbitrary units.


( f ) Representative WB analysis of IGF2R expression in sarcolemma protein lysates of the TA and VM muscles of healthy , DMD , C57Bl6 / J and mdx ( n = 2 per healthy and DMD ; n = 10 mice per group ) . Densitometry analysis of IGF2R expression , results are shown as a fold increase , as indicated in the lower panel in arbitrary units . One - way ANOVA . * p < 0 . 05 ; * * * * p < 0 . 0001 . All values are expressed as the mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative WB analysis of IGF2R expression in sarcolemma protein lysates of the TA and VM muscles of healthy, DMD, C57Bl6/J and mdx (n=2 per healthy and DMD; n=10 mice per group). Densitometry analysis of IGF2R expression, results are shown as a fold increase, as indicated in the lower panel in arbitrary units. One-way ANOVA. *p<0.05; ****p<0.0001. All values are expressed as the mean±SEM.


Quantification of the relative frequency of myofibre cross - sectional areas ( CSAs ) expressed as a frequency distribution in the TA and VM muscles of untreated mdx mice or mdx mice treated with a low or high dose of anti - IGF2R for 4 and 9 weeks ( a ) .

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

Quantification of the relative frequency of myofibre cross-sectional areas (CSAs) expressed as a frequency distribution in the TA and VM muscles of untreated mdx mice or mdx mice treated with a low or high dose of anti-IGF2R for 4 and 9 weeks (a).


( b ) Quantification of regenerating myofibres as a proportion of centrally nucleated fibres in the total number of fibres after anti - IGF2R administration . The quantification of regenerating and M - cadherin - positive satellite cells per section is shown . One - way ANOVA test , * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of regenerating myofibres as a proportion of centrally nucleated fibres in the total number of fibres after anti-IGF2R administration. The quantification of regenerating and M-cadherin-positive satellite cells per section is shown. One-way ANOVA test, **p<0.01; ***p<0.001; ****p<0.0001.


( c ) Representative WB analysis of neonatal myosin heavy chain ( neonatal MHC ) expression and quantification expressed as the ratio of neonatal MHC / β - actin in untreated mdx mice and mdx mice treated with low and high doses of anti - IGF2R for 4 and 9 weeks . One - way ANOVA . * p < 0 . 05 ; * * * p < 0 . 0001 ( n = 10 mice per group ) .

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

(c) Representative WB analysis of neonatal myosin heavy chain (neonatal MHC) expression and quantification expressed as the ratio of neonatal MHC/β-actin in untreated mdx mice and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; ***p<0.0001 (n=10 mice per group).


( d ) Specific force ( maximal tetanic force normalized to cross - sectional area , Po / CSA ) from TA and VM muscles in C57Bl6 / J mice , untreated mdx mice , and mdx mice treated with low and high doses of anti - IGF2R for 4 and 9 weeks . One - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ( n = 10 mice per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Specific force (maximal tetanic force normalized to cross-sectional area, Po/CSA) from TA and VM muscles in C57Bl6/J mice, untreated mdx mice, and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001 (n=10 mice per group).


SERCA activity quantified as ATPase hydrolysis activity vs . min ( e ) in C57Bl6 / J mice , untreated mdx mice , and mdx mice treated with low and high doses of anti - IGF2R for 4 and 9 weeks . One - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ( n = 10 mice per group ) .

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

SERCA activity quantified as ATPase hydrolysis activity vs. min (e) in C57Bl6/J mice, untreated mdx mice, and mdx mice treated with low and high doses of anti-IGF2R for 4 and 9 weeks. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001 (n=10 mice per group).


( a ) Representative WB analysis of active 48 kDa and inactive 60 kDa calcineurin A and β - actin expression in the TA and VM muscles in untreated C57BL mice , untreated mdx mice and mdx treated with low and high doses of anti - IGF2R for 4 and 9 weeks . Densitometry analysis of data expressed as ratios of 48 kDa and 60 kDa calcineurin A / β - actin in arbitrary units . Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ( n = 10 mice per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 WB analysis of active 48 kDa and inactive 60 kDa calcineurin A and β-actin expression in the TA and VM muscles in untreated C57BL mice, untreated mdx mice and mdx treated with low and high doses of anti-IGF2R for 4 and 9 weeks. Densitometry analysis of data expressed as ratios of 48 kDa and 60 kDa calcineurin A/β-actin in arbitrary units. Two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001 (n=10 mice per group).


( b ) Representative WB analysis of CAMKII , p - CAMKII and GAPDH expression quantified and shown as CAMKII / GAPDH and p - CAMKII / CAMKII ratios in arbitrary units . Two - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ( n = 10 mice per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 WB analysis of CAMKII, p-CAMKII and GAPDH expression quantified and shown as CAMKII/GAPDH and p-CAMKII/CAMKII ratios in arbitrary units. Two-way ANOVA. *p<0.05; **p<0.01 (n=10 mice per group).


( c ) Representative WB analysis of PKCα , p - PKCα and β - actin expression levels quantified and expressed as the PKCα / β - actin and p - PKCα / PKCα ratios in arbitrary units ( n = 10 mice per group ) . All values are expressed as the mean ± SEM .

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

(c) Representative WB analysis of PKCα, p-PKCα and β-actin expression levels quantified and expressed as the PKCα/β-actin and p-PKCα/PKCα ratios in arbitrary units (n=10 mice per group). All values are expressed as the mean±SEM.


WB analysis performed in TA and VM muscles of mdx mice treated with anti - IGF2R using antibodies against CD20 , pSer + pThr and vinculin . Densitometry analysis of data expressed as the pSer + pThr / CD20 and CD20 / vinculin ratio in arbitrary units . One - way ANOVA * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 for the comparison with untreated mice ( n = 10 mice per group ) ( a ) .

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

WB analysis performed in TA and VM muscles of mdx mice treated with anti-IGF2R using antibodies against CD20, pSer+pThr and vinculin. Densitometry analysis of data expressed as the pSer+pThr/CD20 and CD20/vinculin ratio in arbitrary units. One-way ANOVA *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for the comparison with untreated mice (n=10 mice per group)(a).


Representative WB analysis performed on TA and VM muscles of mdx mice treated with anti - IGF2R using anti - IGF1R , anti - pospho - IGF1R and GAPDH . Densitometry analysis of data expressed as the pIGF1R / IGF1R ratio normalized to GAPDH content in arbitrary units . One - way ANOVA . * * * * p < 0 . 0001 for the comparison with untreated mice ( n = 10 mice per group ) ( b ) . All values are expressed as the mean ± SEM .

Representative WB analysis performed on TA and VM muscles of mdx mice treated with anti-IGF2R using anti-IGF1R, anti-pospho-IGF1R and GAPDH. Densitometry analysis of data expressed as the pIGF1R/IGF1R ratio normalized to GAPDH content in arbitrary units. One-way ANOVA. ****p<0.0001 for the comparison with untreated mice (n=10 mice per group) (b). All values are expressed as the mean±SEM.


Corrosion casting results showing the linearization of capillaries in IGF2R - treated VM mdx muscles . Pericytes around capillaries are shown in the insert box ( a ) . Scale bars = 100μm .

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

Corrosion casting results showing the linearization of capillaries in IGF2R-treated VM mdx muscles. Pericytes around capillaries are shown in the insert box (a). Scale bars=100μm.


Representative immunofluorescence results ( b ) of longitudinal sections of VM mdx muscles for the CD146 - and NG2 - positive capillaries are shown .

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

Representative immunofluorescence results (b) of longitudinal sections of VM mdx muscles for the CD146- and NG2-positive capillaries are shown.


Representative immunofluorescence quantification ( c ) of longitudinal sections of VM mdx muscles for the CD146 - and NG2 - positive capillaries are shown . Scale bars = 75μm . One - way ANOVA . * * * * p < 0 . 0001 for the comparison with untreated mice ( n = 10 mice per group ) . All values are expressed as the mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 quantification (c) of longitudinal sections of VM mdx muscles for the CD146- and NG2-positive capillaries are shown. Scale bars=75μm. One-way ANOVA. ****p<0.0001 for the comparison with untreated mice (n=10 mice per group). All values are expressed as the mean± SEM.


( d ) Representative WB showing CD146 and NG2 expression in total protein lysates of TA and VM muscles of mdx mice treated with anti - IGF2R . Densitometry analysis of data expressed as the ratio of the indicated antibody / β - actin in arbitrary units . One - way ANOVA . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 for the comparison with untreated mice ( n = 10 mice per group ) . All values are expressed as the mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 WB showing CD146 and NG2 expression in total protein lysates of TA and VM muscles of mdx mice treated with anti-IGF2R. Densitometry analysis of data expressed as the ratio of the indicated antibody/β-actin in arbitrary units. One-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for the comparison with untreated mice (n=10 mice per group). All values are expressed as the mean ± SEM.


( e ) Representative Ve - cadherin ( red ) and CD31 ( green ) expression in small vessels of VM muscles from untreated mdx and mdx treated with low dose of anti - IGF2R for 4 weeks . Scale bars = 25 μm .

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(e) Representative Ve-cadherin (red) and CD31 (green) expression in small vessels of VM muscles from untreated mdx and mdx treated with low dose of anti-IGF2R for 4 weeks. Scale bars=25 μm.


Representative grey - scale images of primary rat hippocampal neurons ( DIV 10 ) transfected with GFP and the indicated miRNA mimics . Quantification of the mean number of intersections by Sholl analysis . GFP - only transfected conditions were set to one in each experiment .

O O O O O O O O O O O O O O O O O O O O O O O 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 grey-scale images of primary rat hippocampal neurons (DIV 10) transfected with GFP and the indicated miRNA mimics. Quantification of the mean number of intersections by Sholl analysis. GFP-only transfected conditions were set to one in each experiment.


Representative grey - scale images of GFP - transfected Cacna1c + / + ( WT ) or Cacna1c + / - primary rat hippocampal neurons ( DIV 10 ) . Scale bars = 50 μm . Quantification of the mean number of intersections by Sholl analysis . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 3 independent experiments ; Unpaired two - sample t - Test , * * p = 0 . 0096 ) . Fold changes represent changes in dendritic complexity relative to the control condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 grey-scale images of GFP-transfected Cacna1c+/+ (WT) or Cacna1c+/- primary rat hippocampal neurons (DIV 10). Scale bars = 50 μm.   Quantification of the mean number of intersections by Sholl analysis. Data are represented as scattered dot plots with bar, mean±S.D. (n=3 independent experiments; Unpaired two-sample t-Test, **p=0.0096). Fold changes represent changes in dendritic complexity relative to the control condition.


Representative images of DIV 10 hippocampal neurons co - transfected with control or miR - 499 - 5p mimics and a pMT2 - CACNB2 expression plasmid ( CACNB2 ) . Scale bars = 50 μm . Quantification of the mean number of intersections by Sholl analysis . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 3 independent experiments ; Two - way ANOVA : no main effect of the CACNB2 transfection p = 0 . 5027 or the miRNA mimics p = 0 . 0805 , main effect of the CACBN2 transfection by miRNA mimics interaction p = 0 . 0213 , Tukey ' s HSD : * p = 0 . 0303 ) . Fold changes represent the changes in dendritic complexity of transfected neurons compared to non - treated control neurons . ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 DIV 10 hippocampal neurons co-transfected with control or miR-499-5p mimics and a pMT2-CACNB2 expression plasmid (CACNB2). Scale bars = 50 μm.   Quantification of the mean number of intersections by Sholl analysis. Data are represented as scattered dot plots with bar, mean±S.D. (n=3 independent experiments; Two-way ANOVA: no main effect of the CACNB2 transfection p=0.5027 or the miRNA mimics p=0.0805, main effect of the CACBN2 transfection by miRNA mimics interaction p=0.0213, Tukey's HSD: *p=0.0303). Fold changes represent the changes in dendritic complexity of transfected neurons compared to non-treated control neurons. ns = not significant.


A ) Representative images of DIV 19 rat hippocampal neurons co - transfected with GFP ( green channel ) and Cav1 . 2 - HA ( red channel ) , together with either control or miR - 499 - 5p mimics . After 12 - 13 days of expression , labeling with Anti - HA antibodies was performed in live conditions to identify cell surface Cav1 . 2 channels . Whole cells : scale bars = 20 μm ; cell body and dendrite insets : scale bars = 5 μm . B ) Quantification of the area occupied by surface Cav1 . 2 fluorescence normalized to the total cell area ( GFP fluorescence ) from non - permeabilized neurons transfected as in A ) . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 4 independent experiments ; Paired two - sample t - Test * p = 0 . 0318 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of DIV 19 rat hippocampal neurons co-transfected with GFP (green channel) and Cav1.2-HA (red channel), together with either control or miR-499-5p mimics. After 12-13 days of expression, labeling with Anti-HA antibodies was performed in live conditions to identify cell surface Cav1.2 channels. Whole cells: scale bars = 20 μm; cell body and dendrite insets: scale bars= 5 μm. B) Quantification of the area occupied by surface Cav1.2 fluorescence normalized to the total cell area (GFP fluorescence) from non-permeabilized neurons transfected as in A). Data are represented as scattered dot plots with bar, mean±S.D. (n=4 independent experiments; Paired two-sample t-Test *p=0.0318).


D ) Representative Western blot images of CACNB2 ( upper panel ) and GAPDH ( lower panel ) protein expression levels in the hippocampus of WT rats and Cacna1c + / - rats injected with the AAV - Control or AAV - miR - 499 hairpin viruses . GAPDH was used as a loading control E ) Quantification of CACNB2 Western blots using hippocampal protein lysate from wild type rats injected with either AAV - control or AAV - miR - 499 . Data are represented as box plot with whiskers and data points ( + : mean , line : median ; whiskers : minimum and maximum values ) . ( * p = 0 . 0103 , Unpaired two - sample t - Test ; n = 7 rats in the AAV - Control group , n = 6 rats in the AAV - miR - 499 group ) . F ) Quantification of CACNB2 Western blots using hippocampal protein lysate from Cacna1c + / - rats injected with either AAV - control or AAV - miR - 499 . Data are represented as box plot with whiskers and data points ( + : mean , line : median ; whiskers : minimum and maximum values ) . ( * * p = 0 . 0044 , Unpaired two - sample t - Test ; n = 8 rats in the AAV - Control group , n = 7 rats in the AAV - miR - 499 group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot images of CACNB2 (upper panel) and GAPDH (lower panel) protein expression levels in the hippocampus of WT rats and Cacna1c +/- rats injected with the AAV-Control or AAV-miR-499 hairpin viruses. GAPDH was used as a loading control E) Quantification of CACNB2 Western blots using hippocampal protein lysate from wild type rats injected with either AAV-control or AAV-miR-499. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (*p=0.0103, Unpaired two-sample t-Test; n=7 rats in the AAV-Control group, n=6 rats in the AAV-miR-499 group). F) Quantification of CACNB2 Western blots using hippocampal protein lysate from Cacna1c +/- rats injected with either AAV-control or AAV-miR-499. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (**p=0.0044, Unpaired two-sample t-Test; n=8 rats in the AAV-Control group, n=7 rats in the AAV-miR-499 group).


miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of control ( female = 26 , male = 31 ) , BD ( female = 26 , male = 37 ) , or MDD ( female = 18 , male = 24 ) subjects . Wilcoxon rank sum test after correction for age and antidepressant treatment ( linear model of the form Fold Change ~ Group * Sex + Age + Antidepressant treatment ) . 2 - way ANOVA with correction for sex , age and antidepressant treatment ( linear model of the form Fold Change ~ Group * Sex + Age + Antidepressant treatment ) . Control vs . BD : * * * * p = 8 . 89e - 07 ; control vs . MDD : n . s . p = 0 . 12674 . Data are presented as violin plots with median , quartiles and data points .

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

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 26, male= 31), BD (female= 26, male= 37), or MDD (female= 18, male=24) subjects. Wilcoxon rank sum test after correction for age and antidepressant treatment (linear model of the form Fold Change ~ Group*Sex + Age + Antidepressant treatment). 2-way ANOVA with correction for sex, age and antidepressant treatment (linear model of the form Fold Change ~ Group*Sex + Age + Antidepressant treatment). Control vs. BD: ****p=8.89e-07; control vs. MDD: n.s. p=0.12674. Data are presented as violin plots with median, quartiles and data points.


miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of control ( female = 8 , male = 10 ) and SZ ( female = 8 , male = 15 ) subjects . 2 - way ANOVA , Main effect Sex : p = 0 . 9785 ; main effect Group : p = 0 . 0924 ; main effect Interaction : p = 0 . 9802 . Tukey ' s HSD : p = 0 . 0924 , ns . Data are presented as violin plots with median , quartiles and data points .

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

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 8, male= 10) and SZ (female= 8, male= 15) subjects. 2-way ANOVA, Main effect Sex: p=0.9785; main effect Group: p= 0.0924; main effect Interaction: p= 0.9802. Tukey's HSD: p=0.0924, ns. Data are presented as violin plots with median, quartiles and data points.


miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of control ( female = 26 , male = 31 ) , BD I ( female = 9 , male = 9 ) , or BDI II ( female = 12 , male = 16 ) subjects . 2 - way ANOVA , Main effect Sex : p = 0 . 174 ; main effect Group : * * * * p = 4 . 07e - 09 ; Interaction Group x Sex : p = 0 . 311 . Tukey ' s HSD : control vs . BDI : * * * * p = 0 . 0000037 . control vs . BDII : p = 0 . 0000002 . Data are presented as violin plots with median , quartiles and data points .

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

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 26, male= 31), BD I (female= 9, male= 9), or BDI II (female= 12, male=16) subjects. 2-way ANOVA, Main effect Sex: p=0.174; main effect Group: ****p=4.07e-09; Interaction Group x Sex: p=0.311. Tukey's HSD: control vs. BDI: ****p=0.0000037. control vs. BDII:p=0.0000002. Data are presented as violin plots with median, quartiles and data points.


miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of control subjects ( female = 26 , male = 31 ) , and BD subjects in different mood states ( depressive ( female = 7 , male = 11 ) , euthymic ( female = 7 , male = 4 ) , hypomanic ( female = 2 , male = 6 ) , manic ( female = 2 , male = 1 ) , mixed ( female = 1 , male = 1 ) . 2 - way ANOVA , main effect Sex : p = 0 . 260 ; main effect Group : p = 1 . 26e - 07 ; Interaction Group x Sex : p = 0 . 059 ; Tukey ' s HSD : control vs . depressive * * * p = 0 . 0002621 , control vs . euthymic * * * p = 0 . 0008533 , control vs . hypomanic * * * * p = 0 . 0000470 . Data are presented as violin plots with median , quartiles and data points .

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

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control subjects (female= 26, male= 31), and BD subjects in different mood states (depressive (female= 7, male=11), euthymic (female= 7, male=4), hypomanic (female=2, male=6), manic (female=2, male=1), mixed (female=1, male=1). 2-way ANOVA, main effect Sex: p=0.260; main effect Group: p=1.26e-07; Interaction Group x Sex: p=0.059; Tukey's HSD: control vs. depressive ***p=0.0002621, control vs. euthymic ***p=0.0008533, control vs. hypomanic ****p=0.0000470. Data are presented as violin plots with median, quartiles and data points.


miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of healthy control subjects ( - CMT , n = 17 ) and healthy subjects with a history of childhood maltreatment ( + CMT , n = 17 ) . ( * * * * p < 0 . 0001 , Mann - Whitney U test ) . CMT : Childhood maltreatment . Data are presented as violin plots with median , quartiles and data points .

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

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of healthy control subjects (-CMT, n=17) and healthy subjects with a history of childhood maltreatment (+CMT, n=17). (****p<0.0001, Mann-Whitney U test). CMT: Childhood maltreatment. Data are presented as violin plots with median, quartiles and data points.


Association of miR - 499 - 5p expression and GMV in HC ( n = 23 ) : miR - 499 - 5p levels were negatively correlated with a GMV cluster comprising parts of the left Wernicke language area ( i . e . transverse temporal gyrus , the left parietal operculum and the left superior temporal gyrus ) ( in blue ) at p < . 05 cluster - level family wise error - corrected ( FWE ) for multiple comparisons after an initial threshold of p < . 001 uncorrected ( k = 1090 , x / y / z = - 42 / - 30 / 15 , t = 4 . 7 , p = . 045 ) .

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

Association of miR-499-5p expression and GMV in HC (n=23): miR-499-5p levels were negatively correlated with a GMV cluster comprising parts of the left Wernicke language area (i.e. transverse temporal gyrus, the left parietal operculum and the left superior temporal gyrus) (in blue) at p<.05 cluster-level family wise error-corrected (FWE) for multiple comparisons after an initial threshold of p<.001 uncorrected (k=1090, x/y/z=-42/-30/15, t= 4.7, p=.045).


( B ) Plaque formation was monitored by automated scanning Levin - Reisman et al . , 2010 ( ) on a lawn of infected PY79 ( WT ) ( 10 - 6 PFU / ml ) cells spread over an MB agar plate . Shown are images captured at the indicated time points ( hrs ) . Scale bar 2 mm . Corresponds to Movie EV2 . ( C - D ) The dynamic of SPP1 ( C ) and Phi29 ( D ) plaque formation following PY79 ( WT ) infection was monitored as described in ( B ) . Shown is the diameter of individual plaques for each phage ( n ≥ 12 ) , with the average highlighted in red .

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

(B) Plaque formation was monitored by automated scanning Levin-Reisman et al., 2010() on a lawn of infected PY79 (WT) (10-6 PFU/ml) cells spread over an MB agar plate. Shown are images captured at the indicated time points (hrs). Scale bar 2 mm. Corresponds to Movie EV2. (C-D) The dynamic of SPP1 (C) and Phi29 (D) plaque formation following PY79 (WT) infection was monitored as described in (B). Shown is the diameter of individual plaques for each phage (n ≥ 12), with the average highlighted in red.


( A ) The indicated strains were infected with SPP1 ( 10 - 6 PFU / ml ) , spread over MB plates , and plaque diameter was monitored after 20 hrs of incubation . Shown is average plaque diameter and SD for each strain ( n ≥ 54 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The indicated strains were infected with SPP1 (10-6 PFU/ml), spread over MB plates, and plaque diameter was monitored after 20 hrs of incubation. Shown is average plaque diameter and SD for each strain (n ≥ 54).


( D ) PY79 ( WT ) and ET19 ( ∆ sigX ) cells were infected with SPP1 at either high ( phages : bacteria 1 : 1 ) or low ( 1 : 20 ) MOI , and OD600nm was followed at 2 min intervals . Shown is a representative experiment out of 6 biological repeats , and the average values and SD of 8 technical repeats .

(D) PY79 (WT) and ET19 (∆sigX) cells were infected with SPP1 at either high (phages:bacteria 1:1) or low (1:20) MOI, and OD600nm was followed at 2 min intervals. Shown is a representative experiment out of 6 biological repeats, and the average values and SD of 8 technical repeats.


( E ) BDR2637 ( Pveg - mCherry ) ( WT , purple ) and ET191 ( PrrnE - gfp , ∆ sigX ) ( ∆ sigX , cyan ) cells were mixed , infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad and plaque formation was followed by time lapse confocal microscopy . Shown are overlay images from mCherry ( purple ) and GFP ( cyan ) signals of the bacterial lawn captured at the indicated time points ( hrs ) . The plaque is seen as a hole formed on the bacterial lawn . Scale bar 100 µm . Corresponds to Movie EV4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BDR2637 (Pveg-mCherry) (WT, purple) and ET191 (PrrnE-gfp, ∆sigX) (∆sigX, cyan) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from mCherry (purple) and GFP (cyan) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm. Corresponds to Movie EV4.


( F ) ET27 ( PsigX - gfp ) cells were infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad , and plaque formation was followed by time lapse confocal microscopy . Shown are fluorescence from GFP ( upper panels ) and corresponding phase contrast images ( lower panels ) , captured at the indicated time points ( hrs ) . The plaque is seen as a hole formed on the bacterial lawn . Scale bar 100 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 O O O O O O O O O O O O O O O O O O O O O

(F) ET27 (PsigX-gfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluorescence from GFP (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.


( C ) ET26 ( PsigX - sigX - gfp ) cells were visualized by fluorescence microscopy . Shown are signal from SigX - GFP ( cyan ) ( left panel ) , and an overlay image of phase contrast ( grey ) and signal from SigX - GFP ( cyan ) ( right panel ) . Scale bar 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

(C) ET26 (PsigX-sigX-gfp) cells were visualized by fluorescence microscopy. Shown are signal from SigX-GFP (cyan) (left panel), and an overlay image of phase contrast (grey) and signal from SigX-GFP (cyan) (right panel). Scale bar 1 μm.


( D ) BDR2637 ( Pveg - mCherry ) ( WT , purple ) and ET261 ( ∆ yueB , PsigX - sigX - gfp ) ( cyan ) cells were mixed , infected with SPP1 at 5 : 1 ( phages : bacteria ) MOI , placed on an agarose pad and followed by time lapse fluorescence microscopy . Shown are overlay images from mCherry ( purple ) , SigX - GFP ( cyan ) , and phase contrast ( grey ) , captured at the indicated time points post infection . Scale bar 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 O O O O O 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) BDR2637 (Pveg-mCherry) (WT, purple) and ET261 (∆yueB, PsigX-sigX-gfp) (cyan) cells were mixed, infected with SPP1 at 5:1 (phages:bacteria) MOI, placed on an agarose pad and followed by time lapse fluorescence microscopy. Shown are overlay images from mCherry (purple), SigX-GFP (cyan), and phase contrast (grey), captured at the indicated time points post infection. Scale bar 1 μm.


( B ) ET29 ( Pveg - mCherry , PIPTG - sigX ) ( purple ) cells were grown in the presence of IPTG and mixed with PY79 ( WT ) cells . The mixture was infected with SPP1 - lysin - yfp 5 : 1 ( phages : bacteria ) MOI , placed on an IPTG - containing agarose pad , and followed by time lapse fluorescence microscopy . Shown are overlay images of phase contrast ( grey ) , signal from mCherry labeled cells ( purple ) , and signal from Lysin - SPP1 - YFP ( cyan ) , captured at the indicated time points post infection ( upper panels ) . Corresponding signal from Lysin - SPP1 - YFP ( cyan ) is shown separately ( lower panels ) . Arrows highlight the delayed infection of ET29 cells . Scale bar 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) ET29 (Pveg-mCherry, PIPTG-sigX) (purple) cells were grown in the presence of IPTG and mixed with PY79 (WT) cells. The mixture was infected with SPP1-lysin-yfp 5:1 (phages:bacteria) MOI, placed on an IPTG-containing agarose pad, and followed by time lapse fluorescence microscopy. Shown are overlay images of phase contrast (grey), signal from mCherry labeled cells (purple), and signal from Lysin-SPP1-YFP (cyan), captured at the indicated time points post infection (upper panels). Corresponding signal from Lysin-SPP1-YFP (cyan) is shown separately (lower panels). Arrows highlight the delayed infection of ET29 cells. Scale bar 1 μm.


( E ) OF83 ( Ppen - lacIΔ11 - cfp ) ( WT ) and ET40 ( Ppen - lacIΔ11 - cfp , PIPTG - sigX ) cells , grown in the presence or absence of IPTG , were infected with SPP1 - delX110lacO64 at 5 : 1 ( phages : bacteria ) MOI . The formation of LacI - CFP foci on phage DNA was monitored 10 min post infection . Non - infected OF83 cells were used for comparison . Shown are overlay images of phase contrast ( grey ) and signal from LacI - CFP ( cyan ) . Scale bar 1 µm . ( F ) Quantification of the experiment described in ( E ) . Shown is the percentage of LacI - CFP foci 10 min post infection of OF83 and ET40 cells by SPP1 , with average values and SD ( n ≥ 850 cells for each population ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) OF83 (Ppen-lacIΔ11-cfp) (WT) and ET40 (Ppen-lacIΔ11-cfp, PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1-delX110lacO64 at 5:1 (phages:bacteria) MOI. The formation of LacI-CFP foci on phage DNA was monitored 10 min post infection. Non-infected OF83 cells were used for comparison. Shown are overlay images of phase contrast (grey) and signal from LacI-CFP (cyan). Scale bar 1 µm. (F) Quantification of the experiment described in (E). Shown is the percentage of LacI-CFP foci 10 min post infection of OF83 and ET40 cells by SPP1, with average values and SD (n ≥ 850 cells for each population).


( A ) ET28 ( PIPTG - sigX ) and ET42 ( ∆ dltA , PIPTG - sigX ) cells , grown in the presence or absence of IPTG , were infected with SPP1 at 1 : 20 ( phages : bacteria ) MOI , and OD600nm was followed at 2 min intervals . Shown is a representative experiment out of 6 biological repeats , with the average values and SD of 8 technical repeats .

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

(A) ET28 (PIPTG-sigX) and ET42 (∆dltA, PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1 at 1:20 (phages:bacteria) MOI, and OD600nm was followed at 2 min intervals. Shown is a representative experiment out of 6 biological repeats, with the average values and SD of 8 technical repeats.


( D ) PY79 ( WT ) and ET41 ( ΔdltA ) cells were infected with SPP1 ( 10 - 6 PFU / ml ) , spred over an MB agar plate , and plaque diameter was monitored after 20 hrs of incubation . Shown is plaque diameter distributions for each strain ( n ≥ 40 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PY79 (WT) and ET41 (ΔdltA) cells were infected with SPP1 (10-6 PFU/ml), spred over an MB agar plate, and plaque diameter was monitored after 20 hrs of incubation. Shown is plaque diameter distributions for each strain (n ≥ 40).


( F ) AR16 ( PrrnE - gfp ) ( WT , cyan ) and ET411 ( Pveg - mCherry , ∆ dltA ) ( ∆ dltA , purple ) cells were mixed , infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad , and plaque formation was followed by time lapse confocal microscopy . Shown are overlay images from GFP ( cyan ) and mCherry ( purple ) signals of the bacterial lawn captured at the indicated time points ( hrs ) . The plaque is seen as a hole formed on the bacterial lawn . Scale bar 150 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 O O O O O O O O O O O O O O O O O O O O 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) AR16 (PrrnE-gfp) (WT, cyan) and ET411 (Pveg-mCherry, ∆dltA) (∆dltA, purple) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from GFP (cyan) and mCherry (purple) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 150 µm.


( G ) ET43 ( dltA - yfp ) cells were infected with low concentrations ( 10 - 8 PFU / ml ) of SPP1 , placed on an agarose pad , and plaque formation was followed by time lapse confocal microscopy . Shown are fluoresce from DltA - YFP signal ( upper panels ) and corresponding phase contrast images ( lower panels ) , captured at the indicated time points ( hrs ) . The plaque is seen as a hole formed on the bacterial lawn . Scale bar 100 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 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) ET43 (dltA-yfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluoresce from DltA-YFP signal (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.


A , Representative images of controls from the siRNA screen for HU - induced RPA accumulation . GFP - RPA1 and RFP - PCNA reporter cells were transfected with siRNAs and 48 hrs later treated with HU ( 3 mM ) for 2 hrs prior to pre - extraction , fixation and imaging .

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

A, Representative images of controls from the siRNA screen for HU-induced RPA accumulation. GFP-RPA1 and RFP-PCNA reporter cells were transfected with siRNAs and 48 hrs later treated with HU (3 mM) for 2 hrs prior to pre-extraction, fixation and imaging.


B , Gene ranking based on siRNA scores from two independent screens . 219 genes were targeted by three individual siRNAs and ranked based on P - values derived from RSA and Fisher ' s analysis . Selected hits are highlighted .

O O O O O O O O O O O O O O O O O O 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, Gene ranking based on siRNA scores from two independent screens. 219 genes were targeted by three individual siRNAs and ranked based on P-values derived from RSA and Fisher's analysis. Selected hits are highlighted.


C , Validation of selected genes by single cell analysis of GFP - RPA1 intensity in pre - extracted cells . Only S - phase cells positive for RFP - PCNA were analysed . a , b , c denote independent siRNAs ; Ctrl , Control . Median with interquartile range of relative intensity per cell is shown , n > 4000 . Mann - Whitney : * * * p < 10 - 4 , n . s . non - significant . One representative experiment out of two biological replicas is shown .

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

C, Validation of selected genes by single cell analysis of GFP-RPA1 intensity in pre-extracted cells. Only S-phase cells positive for RFP-PCNA were analysed. a, b, c denote independent siRNAs; Ctrl, Control. Median with interquartile range of relative intensity per cell is shown, n > 4000. Mann-Whitney: *** p < 10-4, n.s. non-significant. One representative experiment out of two biological replicas is shown.


D , Analysis of DNA synthesis rate . Cells were pulsed 15 min with EdU and EdU intensity was analysed in S - phase cells positive for RFP - PCNA . EdU incorporation is shown relative to siCtrl .

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

D, Analysis of DNA synthesis rate. Cells were pulsed 15 min with EdU and EdU intensity was analysed in S-phase cells positive for RFP-PCNA. EdU incorporation is shown relative to siCtrl.


A , DNA replication measured by EdU incorporation . RPF - PCNA reporter cells were transfected with indicated siRNAs and pulsed with EdU for 20 min . S - phase cells positive for RFP - PCNA were analysed . n > 150 . Error bars , SD ; n = 3 biological replicas .

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

A, DNA replication measured by EdU incorporation. RPF-PCNA reporter cells were transfected with indicated siRNAs and pulsed with EdU for 20 min. S-phase cells positive for RFP-PCNA were analysed. n > 150. Error bars, SD; n= 3 biological replicas.


B , Complementation analysis of DNA synthesis . Stable cell lines expressing BRPF3 resistant to the BRPF3 / a siRNA and control ( lacZ - V5 ) were siRNA transfected and analysed for EdU incorporation as in A . Error - bars , SD ; n = 6 biological replicas . Two - tailed t test , * * p < 10 - 2 .

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

B, Complementation analysis of DNA synthesis. Stable cell lines expressing BRPF3 resistant to the BRPF3/a siRNA and control (lacZ-V5) were siRNA transfected and analysed for EdU incorporation as in A. Error-bars, SD; n = 6 biological replicas. Two-tailed t test, ** p<10-2.


C , Comparison of native BRPF1 and BRPF3 complexes purified from K562 cells . ( left ) mass - spec analysis ( spectral counts / total peptides identified ) and ( right ) verification of associated protein by western blot .

O O O O O O O O O O O O O O O O O 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, Comparison of native BRPF1 and BRPF3 complexes purified from K562 cells. (left) mass-spec analysis (spectral counts/total peptides identified) and (right) verification of associated protein by western blot.


D , Immunoprecipitation analysis of BRPF3 deletion mutants . One representative experiment out of two is shown .

D, Immunoprecipitation analysis of BRPF3 deletion mutants. One representative experiment out of two is shown.


E , Histone acetyltransferase assay ( HAT ) with purified BRPF1 / 3 complexes on free histones and mononucleosomes .

E, Histone acetyltransferase assay (HAT) with purified BRPF1/3 complexes on free histones and mononucleosomes.


F , HAT assay with purified BRPF1 / 3 complexes on H3 peptides unmodified or acetylated ( ac ) / methylated ( me ) at K14 and K27 , 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

F, HAT assay with purified BRPF1/3 complexes on H3 peptides unmodified or acetylated (ac)/methylated (me) at K14 and K27, respectively.


G , Analysis of histone acetylation in BRPF3 depleted cells . ( left ) Western blot of siRNA treated U - 2 - OS cells . TSA treatment ( 1 hr ) was included as a positive control . 2x , double amount of extract loaded in 1x . ( right ) H3K14 acetylation levels quantified relative to total H3 . Error bars , SD ; n = 3 biological replicates .

O O 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

G, Analysis of histone acetylation in BRPF3 depleted cells. (left) Western blot of siRNA treated U-2-OS cells. TSA treatment (1 hr) was included as a positive control. 2x, double amount of extract loaded in 1x. (right) H3K14 acetylation levels quantified relative to total H3. Error bars, SD; n = 3 biological replicates.


H , Complementation analysis of H3K14ac . Total cell extracts of siRNA treated lacZ - V5 and BRPF3 - V5 ( siBRPF3 / a resistant ) expressing cells were analysed by western blotting . One representative experiment out of four biological replicas is shown .

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

H, Complementation analysis of H3K14ac. Total cell extracts of siRNA treated lacZ-V5 and BRPF3-V5 (siBRPF3/a resistant) expressing cells were analysed by western blotting. One representative experiment out of four biological replicas is shown.


A - B , Single - molecule analysis of DNA replication by DNA combing of siRNA transfected cells pulse - labelled with IdU ( 10 min ) and CIdU ( 20 min ) . ( A ) Distribution of inter - track distances . Bars represent the median . n > 100 . Mann - Whitney : * * * P < 10 - 3 . One representative experiment out of two biological replicas is shown . ( B ) Size distribution of CldU track length . Bars represent the median . n > 300 . Mann - Whitney : * * * P < 10 - 3 . One representative experiment out of two biological replicas is shown . C , Distribution of intertrack distances measured by IdU / CIdU pulse labelling and DNA fiber analysis . Bars represent the median . n > 110 . Mann - Whitney : * * * * P < 10 - 4 . One representative experiment out of two biological replicas is shown .

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

A-B, Single-molecule analysis of DNA replication by DNA combing of siRNA transfected cells pulse-labelled with IdU (10 min) and CIdU (20 min). (A) Distribution of inter-track distances. Bars represent the median. n > 100. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. (B) Size distribution of CldU track length. Bars represent the median. n > 300. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. C, Distribution of intertrack distances measured by IdU/CIdU pulse labelling and DNA fiber analysis. Bars represent the median. n > 110. Mann-Whitney: **** P < 10-4. One representative experiment out of two biological replicas is shown.


D , Scatter plot of chromatin - bound GFP - RPA1 intensity per cell . GFP - RPA1 and RFP - PCNA reporter cells were transfected with indicated siRNAs and treated 2 hrs with UCN - 01 ( 300 nM ) prior to pre - extraction and fixation . GFP - RPA1 levels were measured in PCNA positive cells . Lines represent medians . n > 150 . t - tests : * * * * P < 10 - 4 , * P < 0 . 05 ; a . u . , arbitrary unit .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Scatter plot of chromatin-bound GFP-RPA1 intensity per cell. GFP-RPA1 and RFP-PCNA reporter cells were transfected with indicated siRNAs and treated 2 hrs with UCN-01 (300 nM) prior to pre-extraction and fixation. GFP-RPA1 levels were measured in PCNA positive cells. Lines represent medians. n > 150. t-tests: **** P < 10-4, * P < 0.05; a.u., arbitrary unit.


E , Analysis of replication factor loading . Soluble proteins from siRNA transfected cells treated as in D where removed by CSK - T extraction and chromatin pellets analysed by western blotting . One representative experiment out of three independent biological replicas is shown . F , Quantification of chromatin - bound MCM2 ( left ) and CDC45 ( right ) from the three independent experiments described in ( E ) . MCM2 and CDC45 levels were normalized to histone H4 levels and shown relative to untreated control . t - tests : * * * P < 10 - 3 , * * P < 0 . 01 , * P < 0 . 05 , n . s . non - significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Analysis of replication factor loading. Soluble proteins from siRNA transfected cells treated as in D where removed by CSK-T extraction and chromatin pellets analysed by western blotting. One representative experiment out of three independent biological replicas is shown. F, Quantification of chromatin-bound MCM2 (left) and CDC45 (right) from the three independent experiments described in (E). MCM2 and CDC45 levels were normalized to histone H4 levels and shown relative to untreated control. t-tests: *** P < 10-3, ** P < 0.01, * P < 0.05, n.s. non-significant.


G , Analysis of chromatin - bound proteins in HBO1 depleted cells as in E . One representative experiment out of two biological replicas is shown .

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

G, Analysis of chromatin-bound proteins in HBO1 depleted cells as in E. One representative experiment out of two biological replicas is shown.


A , ChIP - seq analysis of BRPF3 binding on the human genome . BRPF3 enrichment signal obtained by ChIP - seq with asynchronous ( AS ) , synchronized ( S ) and released into HU ( S + HU ) RKO cells , on the MCM4 ( left ) and Lamin B2 ( right ) replication origins . Signals were compared to the published enrichment signals for ORC1 in HeLa cells ( ( Dellino et al , 2013 ) , GSE37583 ) , HBO1 in RKO cells ( Avvakumov et al . , 2012 , GSE33221 ) , and H3K14ac in IMR90 cells ( GSM521881 ) . The negative control IgG signal shown is from the S + HUchromatin .

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

A, ChIP-seq analysis of BRPF3 binding on the human genome. BRPF3 enrichment signal obtained by ChIP-seq with asynchronous (AS), synchronized (S) and released into HU (S+HU) RKO cells, on the MCM4 (left) and Lamin B2 (right) replication origins. Signals were compared to the published enrichment signals for ORC1 in HeLa cells ((Dellino et al, 2013), GSE37583), HBO1 in RKO cells (Avvakumov et al., 2012, GSE33221), and H3K14ac in IMR90 cells (GSM521881). The negative control IgG signal shown is from the S+HUchromatin.


B , Venn diagrams illustrating the overlap of genome binding sites between ( top ) BRPF3 , HBO1 and ORC1 , and ( bottom ) BRPF3 , BRPF1 / 2 and ORC1 in human cells .

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

B, Venn diagrams illustrating the overlap of genome binding sites between (top) BRPF3, HBO1 and ORC1, and (bottom) BRPF3, BRPF1/2 and ORC1 in human cells.


C , Heatmaps of BRPF3 , HBO1 and H3K14ac ChIP - seq signal on ± 5kb surrounding the TSS of genes . Genes were sorted by BRPF3 abundance from high to low . A . U . arbitrary unit .

O O O O O O O O O O O O O O O O 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, Heatmaps of BRPF3, HBO1 and H3K14ac ChIP-seq signal on ± 5kb surrounding the TSS of genes. Genes were sorted by BRPF3 abundance from high to low. A.U. arbitrary unit.


D , Average profiles of BRPF3 and H3K14ac ChIP - seq signal on ± 2 . 5kb around the centre of ( top ) BRPF3 and ( bottom ) ORC1 genome binding sites .

O O O O O O O O O O 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, Average profiles of BRPF3 and H3K14ac ChIP-seq signal on ± 2.5kb around the centre of (top) BRPF3 and (bottom) ORC1 genome binding sites.


E , ChIP - qPCR analysis of H3K14ac on well - characterized replication origins . H3K14ac enrichments measured relative to IgG control are shown relative to control siRNA treated cells . Error bars , range ; n = 2 biological replicas .

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

E, ChIP-qPCR analysis of H3K14ac on well-characterized replication origins. H3K14ac enrichments measured relative to IgG control are shown relative to control siRNA treated cells. Error bars, range; n= 2 biological replicas.


F , Expression levels of DNA replication factors were not altered by the lack of BRPF3 . Each point represents the expression levels of a gene in control and BRPF3 depleted cells ( GSE65065 ) . Replication factors were classified as described previously ( Alabert et al , 2014 ) . Total genes ( grey ) , DNA replication factors ( colors ) and BRPF3 ( black ) expression levels are shown . Continuous black lines mark 1 . 5 fold change in expression levels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Expression levels of DNA replication factors were not altered by the lack of BRPF3. Each point represents the expression levels of a gene in control and BRPF3 depleted cells (GSE65065). Replication factors were classified as described previously (Alabert et al, 2014). Total genes (grey), DNA replication factors (colors) and BRPF3 (black) expression levels are shown. Continuous black lines mark 1.5 fold change in expression levels.


A , Western blot of DNA damage signalling in BRPF3 depleted cells . siRNA transfected U - 2 - OS cells were treated with HU for 2 h or 24 h . One representative experiment out of two biological experiments is shown .

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

A, Western blot of DNA damage signalling in BRPF3 depleted cells. siRNA transfected U-2-OS cells were treated with HU for 2 h or 24 h. One representative experiment out of two biological experiments is shown.


B - C , Recovery from replication stress . siRNA treated cells were treated for 24 hrs with HU ( 0 h ) , and released into normal medium for the indicated times ( see also Fig . S6A , B ) . ( B ) DNA replication rate measured by EdU incorporation . Cells were pulsed with EdU for 15 min and gated as shown in Figure S6A to analyse EdU positive cells . n > 1000 . Mann - Whitney : * * * * P < 10 - 4 . ( C ) Cell cycle progression measured by FACS analysis of DNA content . One representative experiment out of two independent biological replicas is shown .

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

B-C, Recovery from replication stress. siRNA treated cells were treated for 24 hrs with HU (0 h), and released into normal medium for the indicated times (see also Fig. S6A, B). (B) DNA replication rate measured by EdU incorporation. Cells were pulsed with EdU for 15 min and gated as shown in Figure S6A to analyse EdU positive cells. n > 1000. Mann-Whitney: **** P < 10-4. (C) Cell cycle progression measured by FACS analysis of DNA content. One representative experiment out of two independent biological replicas is shown.


( A ) Total lysates from the indicated cell lines cultured in regular conditions at a steady state were subjected to western blotting . s . e . , short exposure ; l . e . , long exposure . The major Merlin band is indicated by an asterisk . The ratio of mono - ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot . The same lysates were also used in Figure EV1A . Asterisks indicate native Merlin , arrows show ubiquitinated Merlin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Total lysates from the indicated cell lines cultured in regular conditions at a steady state were subjected to western blotting. s.e., short exposure; l.e., long exposure. The major Merlin band is indicated by an asterisk. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot. The same lysates were also used in Figure EV1A. Asterisks indicate native Merlin, arrows show ubiquitinated Merlin.


( B ) Total lysates from LN229 cells in which Merlin was or was not knocked out ( KO ) were subjected to western blotting .

O O 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) Total lysates from LN229 cells in which Merlin was or was not knocked out (KO) were subjected to western blotting.


LN229 ( C ) cells were detached ( denoted by sus . , referring to suspension ) by trypsinization ( see Methods ) and reseeded ( attach ) for two ( LN229 ) hours . Total lysates from these cells were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . Ubiquitinated Merlin is marked with arrows .

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

LN229 (C) cells were detached (denoted by sus., referring to suspension) by trypsinization (see Methods) and reseeded (attach) for two (LN229) hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with arrows.


Met5 - A ( D ) cells were detached ( denoted by sus . , referring to suspension ) by trypsinization ( see Methods ) and reseeded ( attach ) for four ( Met5 - A ) hours . Total lysates from these cells were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . Ubiquitinated Merlin is marked with arrows .

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

Met5-A (D) cells were detached (denoted by sus., referring to suspension) by trypsinization (see Methods) and reseeded (attach) for four (Met5-A) hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with arrows.


( E ) LN229 cells transduced with a Merlin or scrambled shRNA were treated with DMSO , ionomycin ( Iono ) or thapsigargin ( TG ) . Total lysates of these cells were subjected to western blotting . The major Merlin band is indicated by an asterisk . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . Ubiquitinated Merlin is marked with an arrow .

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 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

(E) LN229 cells transduced with a Merlin or scrambled shRNA were treated with DMSO, ionomycin (Iono) or thapsigargin (TG). Total lysates of these cells were subjected to western blotting. The major Merlin band is indicated by an asterisk. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with an arrow.


( F ) LN229 cells treated with DMSO or thapsigargin ( TG ) were lysed and subjected to immunoprecipitation with a Merlin antibody or IgG . The immunoprecipitated products were subjected to western blotting . The number of conjugated ubiquitin ( Ub ) moieties was marked . Tetra - ubiquitinated Merlin is indicated by an arrow .

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

(F) LN229 cells treated with DMSO or thapsigargin (TG) were lysed and subjected to immunoprecipitation with a Merlin antibody or IgG. The immunoprecipitated products were subjected to western blotting. The number of conjugated ubiquitin (Ub) moieties was marked. Tetra-ubiquitinated Merlin is indicated by an arrow.


( G ) LN229 cells stably transduced with 6x - Histidine - tagged ubiquitin ( His - Ubi ) were treated with DMSO or thapsigargin ( TG ) . These cells were lysed and subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . Both arrowhead and arrow point to the mono - ubiquitinated Merlin . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

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

(G) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were treated with DMSO or thapsigargin (TG). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Both arrowhead and arrow point to the mono-ubiquitinated Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( H ) LN229 cells stably transduced with 6x - Histidine - tagged ubiquitin ( His - Ubi ) were detached or reseeded as in ( C ) . These cells were lysed and subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . Arrowheads and arrows point to the mono - or di - ubiquitinated Merlin ( see a longer exposed blotting results in Figure EV1D ) . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

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

(H) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were detached or reseeded as in (C). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Arrowheads and arrows point to the mono- or di-ubiquitinated Merlin (see a longer exposed blotting results in Figure EV1D). The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( A ) LN229 cells were detached ( denoted by sus . , referring to suspension ) by trypsinization and reseeded ( denoted by attach ) for two hours . Total lysates from these cells were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LN229 cells were detached (denoted by sus., referring to suspension) by trypsinization and reseeded (denoted by attach) for two hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( B ) LN229 cells treated with DMSO , ionomycin ( Iono ) or thapsigargin ( TG ) were lysed and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

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

(B) LN229 cells treated with DMSO, ionomycin (Iono) or thapsigargin (TG) were lysed and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( C ) LN229 cells treated with DMSO or thapsigargin ( TG ) were subjected to active RAC1 pulldown assay followed by western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

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

(C) LN229 cells treated with DMSO or thapsigargin (TG) were subjected to active RAC1 pulldown assay followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


LN229 cells transduced with vector or EGFP - RAC1 ( Q61L ) ( D ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

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

LN229 cells transduced with vector or EGFP-RAC1(Q61L) (D) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( E ) LN229 cells transduced with vector or Myc - PAK1 ( T423E ) ( E ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

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

(E) LN229 cells transduced with vector or Myc-PAK1(T423E) (E) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( F ) LN229 cells stably transduced with wild - type Merlin or the indicated mutants were treated with DMSO or thapsigargin ( TG ) and analyzed by western blot . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . Native Merlin ( asterisk ) ; ubiquitinated Merlin ( arrow ) .

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

(F) LN229 cells stably transduced with wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG) and analyzed by western blot. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Native Merlin (asterisk); ubiquitinated Merlin (arrow).


( G ) Merlin - depleted LN229 cells stably transduced with Flag - tagged wild - type Merlin or the indicated mutants were treated with DMSO or thapsigargin ( TG ) . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The lysate and immunoprecipitated products were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

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

(G) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( B ) LN229 cells transduced with a pool of four siRNAs against NEDD4L ( + ) or a scrambled siRNA ( - ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot .

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

(B) LN229 cells transduced with a pool of four siRNAs against NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot.


( C ) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

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

(C) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( D ) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were detached by trypsinization ( see Methods ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were detached by trypsinization (see Methods) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( E ) LN229 cells stably transduced with 6x - Histidine - tagged Ubiquitin ( His - Ubi ) were then stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA . These cells were detached as in ( D ) , lysed and subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA. These cells were detached as in (D), lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( F ) HEK293T cells were transfected with indicated genes and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O 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) HEK293T cells were transfected with indicated genes and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( G ) HEK293T cells were transfected with indicated genes . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The immunoprecipitated products were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293T cells were transfected with indicated genes. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( H ) LN229 cells were lysed and subjected to immunoprecipitation with a Merlin antibody , a NEDD4L antibody , or IgG . The immunoprecipitated products were subjected to western blotting .

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

(H) LN229 cells were lysed and subjected to immunoprecipitation with a Merlin antibody, a NEDD4L antibody, or IgG. The immunoprecipitated products were subjected to western blotting.


( A ) LN229 cells transduced with a pool of four siRNAs against AMOTL1 ( + ) or a scrambled siRNA ( - ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

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

(A) LN229 cells transduced with a pool of four siRNAs against AMOTL1 (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( B ) LN229 cells stably transduced with 6x - Histidine - tagged Ubiquitin ( His - Ubi ) were then stably transduced with indicated shRNAs against AMOTL1 or a scrambled shRNA control ( sh - Co . ) . These cells were detached , lysed and subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs against AMOTL1 or a scrambled shRNA control (sh-Co.). These cells were detached, lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( D ) Merlin - depleted LN229 cells stably expressing Flag - HA - tagged Merlin were transduced with scrambled shRNA ( sh - Co . ) or two distinct shRNAs targeting AMOTL1 , treated with DMSO or thapsigargin ( TG ) and subjected to Flag immunoprecipitation followed by western blotting .

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

(D) Merlin-depleted LN229 cells stably expressing Flag-HA-tagged Merlin were transduced with scrambled shRNA (sh-Co.) or two distinct shRNAs targeting AMOTL1, treated with DMSO or thapsigargin (TG) and subjected to Flag immunoprecipitation followed by western blotting.


( E ) HEK293T cells were transfected with the indicated constructs . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The lysate and immunoprecipitated products were subjected to western blotting . PY : PPEY motif ; LY : LPTY motif . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. PY: PPEY motif; LY: LPTY motif. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( F ) Merlin - depleted LN229 cells stably transduced with Flag - tagged wild - type Merlin or the indicated mutants were treated with DMSO or thapsigargin ( TG ) . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The lysate and immunoprecipitated products were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

(F) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( G ) HEK293T cells were transfected with the indicated constructs . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The lysate and immunoprecipitated products were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( A ) LN229 cells stably transduced with Flag - tagged wild - type Merlin or the S518D mutant were treated with thapsigargin and subjected to immunoprecipitation with a Flag antibody and followed by SDS - PAGE . Outlined gel pieces were analyzed by mass spectrometry . ( B ) Numbers of unique peptides of Merlin or Ubiquitin from each outlined gel pieces in ( A ) are shown .

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

(A) LN229 cells stably transduced with Flag-tagged wild-type Merlin or the S518D mutant were treated with thapsigargin and subjected to immunoprecipitation with a Flag antibody and followed by SDS-PAGE. Outlined gel pieces were analyzed by mass spectrometry. (B) Numbers of unique peptides of Merlin or Ubiquitin from each outlined gel pieces in (A) are shown.


( D ) and ( E ) Mass spectrometry spectrums of the indicated peptides from each immunoprecipitated product are shown .

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

(D) and (E) Mass spectrometry spectrums of the indicated peptides from each immunoprecipitated product are shown.


( F ) Merlin - depleted LN229 cells expressing His - Ubiquitin were stably transduced with the indicated HA - tagged Merlin forms or an empty vector . These cells treated with DMSO or thapsigargin were subjected to nickel affinity purification in a denaturing step followed by western blotting for HA . Arrows indicate mono - ubiquitinated Merlin . The red asterisk indicates native Merlin and black asterisks indicate non - specific signals . The ratio of mono - ubiquitinated to native Merlin in each lane of the input blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O 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

(F) Merlin-depleted LN229 cells expressing His-Ubiquitin were stably transduced with the indicated HA-tagged Merlin forms or an empty vector. These cells treated with DMSO or thapsigargin were subjected to nickel affinity purification in a denaturing step followed by western blotting for HA. Arrows indicate mono-ubiquitinated Merlin. The red asterisk indicates native Merlin and black asterisks indicate non-specific signals. The ratio of mono-ubiquitinated to native Merlin in each lane of the input blot was quantified by Image J and is shown under the blot.


( G ) Merlin - depleted LN229 cells were stably transduced with an empty vector , Flag - tagged wild - type ( WT ) Merlin or its K396R mutant . These cells were detached ( S , suspension ) by trypsinization using the procedure described in the Methods section and reseeded ( A , attached ) for two hours . Cell lysates were subjected to immunoprecipitation with a Flag antibody followed by western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Merlin-depleted LN229 cells were stably transduced with an empty vector, Flag-tagged wild-type (WT) Merlin or its K396R mutant. These cells were detached (S, suspension) by trypsinization using the procedure described in the Methods section and reseeded (A, attached) for two hours. Cell lysates were subjected to immunoprecipitation with a Flag antibody followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


( H ) Merlin - depleted LN229 cells stably transduced with wild - type ( WT ) Merlin or its K396R mutant were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

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

(H) Merlin-depleted LN229 cells stably transduced with wild-type (WT) Merlin or its K396R mutant were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( I ) Merlin - depleted LN229 cells stably transduced with HA - tagged wild - type ( WT ) Merlin or its K396R mutant were treated as described in ( G ) and subjected to western blotting . The ratio of mono - ubiquitinated to native null in each lane was quantified by Image J and is shown under the blot .

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

(I) Merlin-depleted LN229 cells stably transduced with HA-tagged wild-type (WT) Merlin or its K396R mutant were treated as described in (G) and subjected to western blotting. The ratio of mono-ubiquitinated to native null in each lane was quantified by Image J and is shown under the blot.


( A ) LN229 cells were treated with DMSO or thapsigargin ( TG ) for the indicated time and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

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

(A) LN229 cells were treated with DMSO or thapsigargin (TG) for the indicated time and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( B ) Merlin - depleted LN229 cells were reconstituted with wild type ( WT ) Merlin or its mutants , treated with DMSO or thapsigargin ( TG ) , and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

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

(B) Merlin-depleted LN229 cells were reconstituted with wild type (WT) Merlin or its mutants, treated with DMSO or thapsigargin (TG), and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( C ) Merlin - depleted LN229 cells were reconstituted with wild type ( WT ) Merlin or its mutants , treated with DMSO or thapsigargin ( TG ) , and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

(C) Merlin-depleted LN229 cells were reconstituted with wild type (WT) Merlin or its mutants, treated with DMSO or thapsigargin (TG), and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( D ) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L ( + ) or a scrambled siRNA ( - ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting .

O O O O O 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

(D) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting.


( E ) Merlin - depleted LN229 cells were stably transduced with empty vector , Flag - tagged wild - type ( WT ) Merlin , or its K396R mutant . These cells were detached ( S , suspension ) by trypsinization using the procedure described in Methods and reseeded ( A , attached ) for two hours . Cell lysates were subjected to western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Merlin-depleted LN229 cells were stably transduced with empty vector, Flag-tagged wild-type (WT) Merlin, or its K396R mutant. These cells were detached (S, suspension) by trypsinization using the procedure described in Methods and reseeded (A, attached) for two hours. Cell lysates were subjected to western blotting.


( F ) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L ( + ) or a scrambled siRNA ( - ) were detached ( Susp . , suspension ) by trypsinization using the procedure described in Methods and reseeded ( Attach , attached ) for two hours . Total lysates from these cells were subjected to western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L (+) or a scrambled siRNA (-) were detached (Susp., suspension) by trypsinization using the procedure described in Methods and reseeded (Attach, attached) for two hours. Total lysates from these cells were subjected to western blotting.


( G ) LN229 cells transfected with indicated siRNAs were seeded for 3 hours . CTGF or CYR61 mRNA in these cells was quantified by q - RT - PCR . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 . Mean ± s . e . m , N = 4 biological repeats , unpaired t - test .

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

(G) LN229 cells transfected with indicated siRNAs were seeded for 3 hours. CTGF or CYR61 mRNA in these cells was quantified by q-RT-PCR. *P<0.05, **P<0.01, ****P<0.0001. Mean±s.e.m, N=4 biological repeats, unpaired t-test.


( A ) LN229 cells stably transduced withHA - Lats1 ( two leftmost panels ) or vector only ( right panel ) were treated with DMSO or thapsigargin ( TG ) and subjected to PLA using HA and Merlin antibodies . Scale bar = 10 μm . ( B ) PLA signals ( dots ) in each cell from the results in ( A ) were quantified . Mean ± s . e . m , two - way ANOVA . * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each data point represents an image field containing an average of 10 cells . N = 4 - 9 images for each condition as indicated . All images were collected from one experiment . Two independent experiments were performed and gave similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LN229 cells stably transduced withHA-Lats1 (two leftmost panels) or vector only (right panel) were treated with DMSO or thapsigargin (TG) and subjected to PLA using HA and Merlin antibodies. Scale bar = 10 μm. (B) PLA signals (dots) in each cell from the results in (A) were quantified. Mean±s.e.m, two-way ANOVA. *P<0.05, ***P<0.001, ****P<0.0001. Each data point represents an image field containing an average of 10 cells. N=4-9 images for each condition as indicated. All images were collected from one experiment. Two independent experiments were performed and gave similar results.


( C ) Merlin - depleted LN229 cells were stably transduced with HA - tagged wild - type ( WT ) Merlin or its mutants . These cells were treated with thapsigargin ( TG ) for 15 minutes and subjected to PLA using HA and Lats1 antibodies . Scale bar = 20 μm . ( D ) PLA signals ( dots ) in each cell from the results in ( C ) were quantified . Mean ± s . e . m , Ordinary one - way ANOVA . * * * * P < 0 . 0001 . Each data point represents an image field containing an average of 10 cells . NVector = 8 , NWT = 16 , NK396R = 18 , NK159R = 17 images . All images were collected from one experiment . Two independent experiments were performed and gave similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Merlin-depleted LN229 cells were stably transduced with HA-tagged wild-type (WT) Merlin or its mutants. These cells were treated with thapsigargin (TG) for 15 minutes and subjected to PLA using HA and Lats1 antibodies. Scale bar = 20 μm. (D) PLA signals (dots) in each cell from the results in (C) were quantified. Mean±s.e.m, Ordinary one-way ANOVA. ****P<0.0001. Each data point represents an image field containing an average of 10 cells. NVector=8, NWT=16, NK396R=18, NK159R=17 images. All images were collected from one experiment. Two independent experiments were performed and gave similar results.


( E ) Merlin - depleted LN229 cells stably transduced with empty vector , Flag - tagged wild - type ( WT ) Merlin , or its K396R mutant were subjected to immunoprecipitation with a Flag antibody followed by western blotting .

O O O O O O O O O O O O O O O O O O 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) Merlin-depleted LN229 cells stably transduced with empty vector, Flag-tagged wild-type (WT) Merlin, or its K396R mutant were subjected to immunoprecipitation with a Flag antibody followed by western blotting.


( F ) HEK293T cells were transfected with indicated proteins and subjected to immunoprecipitation with a Flag antibody . The lysates and immunoprecipitated products were subjected to western blotting .

O O O O O O 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) HEK293T cells were transfected with indicated proteins and subjected to immunoprecipitation with a Flag antibody. The lysates and immunoprecipitated products were subjected to western blotting.


( G ) Merlin - depleted LN229 cells were stably transduced by HA - tagged Merlin . These cells were then transduced with a pool of four siRNAs against NEDD4L or a scrambled control siRNA and treated with DMSO or thapsigargin ( TG ) . PLA was performed in these cells using HA and Lats1 antibodies , and the PLA signals ( dots , showed in Figure EV5E ) in each cell were quantified . Mean ± s . e . m , two - way ANOVA . * * * * P < 0 . 0001 . Each data point represents an image field containing averagely 10 cells . N = 12 images in all conditions , except for Nsi - Control : TG = 13 images . All images were collected from one experiment . Two independent experiments were performed and showed similar results .

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

(G) Merlin-depleted LN229 cells were stably transduced by HA-tagged Merlin. These cells were then transduced with a pool of four siRNAs against NEDD4L or a scrambled control siRNA and treated with DMSO or thapsigargin (TG). PLA was performed in these cells using HA and Lats1 antibodies, and the PLA signals (dots, showed in Figure EV5E) in each cell were quantified. Mean±s.e.m, two-way ANOVA. ****P<0.0001. Each data point represents an image field containing averagely 10 cells. N=12 images in all conditions, except for Nsi-Control:TG=13 images. All images were collected from one experiment. Two independent experiments were performed and showed similar results.


( H ) LN229 cells treated with DMSO or thapsigargin ( TG ) were subjected to cytosolic ( C ) / membrane ( M ) fractionation followed by western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

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

(H) LN229 cells treated with DMSO or thapsigargin (TG) were subjected to cytosolic (C)/membrane (M) fractionation followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


( A ) Met5 - A cells stably transduced with the indicated shRNAs targeting Merlin or a scrambled shRNA were subjected to the BrdU incorporation assay . Scale bar = 40 μm . ( B ) The percentage of BrdU positive cells from ( A ) was quantified . Mean ± s . e . m , N = 3 biological repeats , paired t - test . * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Met5-A cells stably transduced with the indicated shRNAs targeting Merlin or a scrambled shRNA were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (B) The percentage of BrdU positive cells from (A) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.01, ***P<0.001.