( E ) Soluble HttQ25 - CFP degradation is increased after WAC depletion . Anti ‐ WAC , ‐ Actin and ‐ GFP blots after indicated siRNA treatment ( siWAC duplexes ‐ 02 , ‐ 03 , ‐ 04 ) of HttPolyQ ‐ mutant HeLa cell lines and incubation conditions DMSO or DOX ( Doxycyline ) for 3 days . The insoluble fraction was obtained by pelleting the HttQ103 lysates ( see Materials and methods ) . Quantification of normalised average HttQ25 - CFP intensity in DMSO of RISCfree compared with WAC siRNA treatment ; error bar represents s . e . m . ( n = 3 ) : HttQ25 - CFP / actin RF DMSO versus siWAC DMSO , * P = 0 . 0350 ; soluble HttQ103 - CFP / actin RF DMSO versus siWAC DMSO , * P = 0 . 0380 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Soluble HttQ25-CFP degradation is increased after WAC depletion. Anti‐WAC, ‐Actin and ‐GFP blots after indicated siRNA treatment (siWAC duplexes ‐02, ‐03, ‐04) of HttPolyQ‐mutant HeLa cell lines and incubation conditions DMSO or DOX (Doxycyline) for 3 days. The insoluble fraction was obtained by pelleting the HttQ103 lysates (see Materials and methods). Quantification of normalised average HttQ25-CFP intensity in DMSO of RISCfree compared with WAC siRNA treatment; error bar represents s.e.m. (n=3): HttQ25-CFP/actin RF DMSO versus siWAC DMSO, *P=0.0350; soluble HttQ103-CFP/actin RF DMSO versus siWAC DMSO, *P=0.0380.


( C ) Representative image of mESC expressing Nanog - EGFP in + LIF . Bright field , epifluorescence , HILOM , and a max projection image of 2 , 000 frames 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

(C) Representative image of mESC expressing Nanog-EGFP in +LIF. Bright field, epifluorescence, HILOM, and a max projection image of 2,000 frames are shown.


( F ) Confocal images of DRAQ5 staining , showing double stranded DNA ( top ) , immunostaining of H3K27ac ( middle ) , and Nanog - EGFP ( bottom ) , along with the progression of differentiation ( + 2i , + LIF , − LIF , and − LIF 2d ) .

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

(F) Confocal images of DRAQ5 staining, showing double stranded DNA (top), immunostaining of H3K27ac (middle), and Nanog-EGFP (bottom), along with the progression of differentiation (+2i, +LIF, −LIF, and −LIF 2d).


( G ) Swarmplots and violinplots of coefficient of variation of DRAQ5 fluorescence in a nucleus of an mESC expressing Nanog - EGFP at the condition of + 2i ( red , n = 20 cells ) , + LIF ( blue , n = 20 ) , − LIF ( green , n = 23 ) , − LIF + TSA ( light green , n = 22 ) , − LIF2d ( purple , n = 21 ) , and − LIF2d + TSA ( pink , n = 19 ) . Double asterisks and single asterisks indicate p - value < 0 . 05 and 0 . 05 ≤ p - value < 0 . 1 , respectively , in Student ' s t - test .

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

(G) Swarmplots and violinplots of coefficient of variation of DRAQ5 fluorescence in a nucleus of an mESC expressing Nanog-EGFP at the condition of +2i (red, n = 20 cells), +LIF (blue, n = 20), −LIF (green, n = 23), −LIF+TSA (light green, n = 22), −LIF2d (purple, n = 21), and −LIF2d+TSA (pink, n = 19). Double asterisks and single asterisks indicate p-value < 0.05 and 0.05 ≤ p-value < 0.1, respectively, in Student's t-test.


( C ) Typical example of dwell time histograms of Nanog - EGFP obtained within every 400 frames at frame periods of 50 ( green , n = 228 traces ) , 100 ( orange , n = 97 ) , 150 ( red , n = 102 ) , 250 ( magenta , n = 124 ) , and 450 ms ( blue , n = 157 ) in a nucleus in + LIF .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Typical example of dwell time histograms of Nanog-EGFP obtained within every 400 frames at frame periods of 50 (green, n = 228 traces), 100 (orange, n = 97), 150 (red, n = 102), 250 (magenta, n = 124), and 450 ms (blue, n = 157) in a nucleus in +LIF.


( D ) Dwell time histograms of Nanog - EGFP ( left ) and Oct4 - EGFP ( right ) obtained from all cell data ( n = 73 in + 2i , 65 in + LIF , and 66 cells in − LIF for Nanog - EGFP , and n = 57 in + 2i , 53 in + LIF , and 58 cells in − LIF for Oct4 - EGFP ) at frame periods of 50 , 100 , 150 , 250 , and 450 ms in a nucleus in + 2i ( red ) , + LIF ( blue ) , and − LIF conditions ( green ) . Values of koff were obtained by the global fitting approximation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dwell time histograms of Nanog-EGFP (left) and Oct4-EGFP (right) obtained from all cell data (n = 73 in +2i, 65 in +LIF, and 66 cells in −LIF for Nanog-EGFP, and n = 57 in +2i, 53 in +LIF, and 58 cells in −LIF for Oct4-EGFP) at frame periods of 50, 100, 150, 250, and 450 ms in a nucleus in +2i (red), +LIF (blue), and −LIF conditions (green). Values of koff were obtained by the global fitting approximation.


( E ) Histograms of dissociation rate of Nanog - EGFP ( left ) and Oct4 - EGFP ( right ) in an mESC in + 2i ( top ) , + LIF ( middle ) , and − LIF ( bottom ) conditions . Black line shows the fitting result with a normal distribution .

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

(E) Histograms of dissociation rate of Nanog-EGFP (left) and Oct4-EGFP (right) in an mESC in +2i (top), +LIF (middle), and −LIF (bottom) conditions. Black line shows the fitting result with a normal distribution.


( F ) Dependence of expression level of Nanog - EGFP ( upper ) and Oct4 - EGFP ( lower ) on dissociation rate . Considering the distribution of the fluorescent intensity low ( blue ) , middle ( magenta ) , and high ( red ) were defined as cells showing the log value of the fluorescent intensity of < 1 . 9 , 2 . 0 - 2 . 3 , and > 2 . 4 [ a . u . ] , respectively , for Nanog - EGFP or < 1 . 9 , 2 . 0 - 2 . 2 , and < 2 . 3 [ a . u . ] , respectively , for Oct4 - EGFP . Bars and error bars are mean ± SD . For Nanog - EGFP , N = 20 ( high ) , 32 ( middle ) and 20 ( low ) for + 2i , 26 ( high ) , 18 ( middle ) and 21 ( low ) for + LIF , and 20 ( high ) , 26 ( middle ) and 20 cells ( low ) for − LIF . For Oct4 - EGFP , N = 22 ( high ) , 18 ( middle ) and 17 ( low ) for + 2i , 17 ( high ) 16 ( middle ) and 20 ( low ) for + LIF , and 19 ( high ) , 19 ( middle ) and 20 cells ( low ) for − LIF . Asterisks indicate less than 0 . 05 of the p - value in Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dependence of expression level of Nanog-EGFP (upper) and Oct4-EGFP (lower) on dissociation rate. Considering the distribution of the fluorescent intensity low (blue), middle (magenta), and high (red) were defined as cells showing the log value of the fluorescent intensity of < 1.9, 2.0-2.3, and > 2.4 [a.u.], respectively, for Nanog-EGFP or < 1.9, 2.0-2.2, and < 2.3 [a.u.], respectively, for Oct4-EGFP. Bars and error bars are mean ± SD. For Nanog-EGFP, N = 20 (high), 32 (middle) and 20 (low) for +2i, 26 (high), 18 (middle) and 21 (low) for +LIF, and 20 (high), 26 (middle) and 20 cells (low) for −LIF. For Oct4-EGFP, N = 22 (high), 18 (middle) and 17 (low) for +2i, 17 (high) 16 (middle) and 20 (low) for +LIF, and 19 (high), 19 (middle) and 20 cells (low) for −LIF. Asterisks indicate less than 0.05 of the p-value in Student's t-test.


( D , E ) Dependence of expression level of Nanog - EGFP ( left ) and Oct4 - EGFP ( right ) on Rc ( D ) or D ( E ) . Bars and error bars are mean ± SD . For Nanog - EGFP , N = 20 ( high ) , 32 ( middle ) and 20 ( low ) for + 2i , 26 ( high ) , 18 ( middle ) and 21 ( low ) for + LIF , and 20 ( high ) , 26 ( middle ) and 20 cells ( low ) for − LIF . Of Oct4 - EGFP , N = 22 ( high ) , 18 ( middle ) and 17 ( low ) for + 2i , 17 ( high ) 16 ( middle ) and 20 ( low ) for + LIF , and 19 ( high ) , 19 ( middle ) and 20 cells ( low ) for − LIF . Asterisks and double asterisks indicate p - values of less than 0 . 05 and 0 . 01 , respectively , in Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) Dependence of expression level of Nanog-EGFP (left) and Oct4-EGFP (right) on Rc (D) or D (E). Bars and error bars are mean ± SD. For Nanog-EGFP, N = 20 (high), 32 (middle) and 20 (low) for +2i, 26 (high), 18 (middle) and 21 (low) for +LIF, and 20 (high), 26 (middle) and 20 cells (low) for −LIF. Of Oct4-EGFP, N = 22 (high), 18 (middle) and 17 (low) for +2i, 17 (high) 16 (middle) and 20 (low) for +LIF, and 19 (high), 19 (middle) and 20 cells (low) for −LIF. Asterisks and double asterisks indicate p-values of less than 0.05 and 0.01, respectively, in Student's t-test.


( C , D ) The average correlation index g ( r ) of Nanog - EGFP ( C ) and Oct4 - EGFP ( D ) in + 2i ( top ) , + LIF ( middle ) , and − LIF ( bottom ) . Insets are histograms of logarithm value of the intercept g0 ( magenta ) and the exponential decay constant ξ ( orange ) of single mESCs . Black lines in insets show the fitting results with a normal distribution . Error bars represent the standard deviation . N = 72 for + 2i , 56 for + LIF and 56 cells for − LIF of Nanog - EGFP , and N = 57 for + 2i , 53 for + LIF and 58 cells for − LIF of Oct4 - EGFP .

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

(C, D) The average correlation index g(r) of Nanog-EGFP (C) and Oct4-EGFP (D) in +2i (top), +LIF (middle), and −LIF (bottom). Insets are histograms of logarithm value of the intercept g0 (magenta) and the exponential decay constant ξ (orange) of single mESCs. Black lines in insets show the fitting results with a normal distribution. Error bars represent the standard deviation. N = 72 for +2i, 56 for +LIF and 56 cells for −LIF of Nanog-EGFP, and N = 57 for +2i, 53 for +LIF and 58 cells for −LIF of Oct4-EGFP.


( E , F ) Dependence of expression level of Nanog - EGFP ( left ) and Oct4 - EGFP ( right ) on log10g0 ( E ) and log10ξ ( F ) . Error bars represent standard deviations . The definition of low ( blue ) , middle ( magenta ) , and high ( red ) in E and F were the same as that of Fig . 3F . Bars and error bars are mean ± SD . Of Nanog - EGFP , N = 20 ( high ) , 32 ( middle ) and 20 ( low ) for + 2i , 26 ( high ) , 18 ( middle ) and 21 ( low ) for + LIF , and 20 ( high ) , 26 ( middle ) and 20 cells ( low ) for − LIF . Of Oct4 - EGFP , N = 22 ( high ) , 18 ( middle ) and 17 ( low ) for + 2i , 17 ( high ) 16 ( middle ) and 20 ( low ) for + LIF , and 19 ( high ) , 19 ( middle ) and 20 cells ( low ) for − LIF . Asterisks and double asterisks indicate p - values of less than 0 . 05 and 0 . 01 , respectively , in Student ' s t - test . Exclamation marks and double exclamation marks indicate p - values of less than 0 . 05 and 0 . 01 , respectively , in Mann Whitney ' s U - test .

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

(E, F) Dependence of expression level of Nanog-EGFP (left) and Oct4-EGFP (right) on log10g0 (E) and log10ξ (F). Error bars represent standard deviations. The definition of low (blue), middle (magenta), and high (red) in E and F were the same as that of Fig. 3F. Bars and error bars are mean ± SD. Of Nanog-EGFP, N = 20 (high), 32 (middle) and 20 (low) for +2i, 26 (high), 18 (middle) and 21 (low) for +LIF, and 20 (high), 26 (middle) and 20 cells (low) for −LIF. Of Oct4-EGFP, N = 22 (high), 18 (middle) and 17 (low) for +2i, 17 (high) 16 (middle) and 20 (low) for +LIF, and 19 (high), 19 (middle) and 20 cells (low) for −LIF. Asterisks and double asterisks indicate p-values of less than 0.05 and 0.01, respectively, in Student's t-test. Exclamation marks and double exclamation marks indicate p-values of less than 0.05 and 0.01, respectively, in Mann Whitney's U-test.


( B , A typical example of time developments of correlation index g ( r ) ( B ) in a mESC six hours after the removal of 2i and LIF . These were obtained from the same cell .

O O O O O O O O O O O O O O O O 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, A typical example of time developments of correlation index g(r) (B) in a mESC six hours after the removal of 2i and LIF. These were obtained from the same cell.


C ) visualisation of the binding frequency of Nanog - EGFP per unit area ( density ) ( C ) in a mESC six hours after the removal of 2i and LIF . Colour in C corresponds to the density of Nanog dwell events ( events / μm2 ) within 2 , 000 frames . These were obtained from the same cell . Arrows indicate the emergence of hotspots of functioning Nanog , exhibiting obviously higher density than the others .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) visualisation of the binding frequency of Nanog-EGFP per unit area (density) (C) in a mESC six hours after the removal of 2i and LIF. Colour in C corresponds to the density of Nanog dwell events (events/μm2) within 2,000 frames. These were obtained from the same cell. Arrows indicate the emergence of hotspots of functioning Nanog, exhibiting obviously higher density than the others.


( C , D ) Plots of all data of cells categorised with expression level ( red , magenta , and blue ) and with the mean values ( green or cyan ) in each parameter in each condition of Nanog - EGFP ( C ) or Oct4 - EGFP ( D ) . The definition of low ( blue ) , middle ( magenta ) , and high ( red ) in + 2i , + LIF , and − LIF were the same as that of Fig . 3F , and those in − LIF2d were the same as B in this figure . Error bars represent standard deviations . For Nanog - EGFP , N = 20 ( high ) , 32 ( middle ) and 20 ( low ) for + 2i , 26 ( high ) , 18 ( middle ) and 21 ( low ) for + LIF , 20 ( high ) , 26 ( middle ) and 20 cells ( low ) for − LIF , and 20 ( high ) , 20 ( middle ) and 19 cells ( low ) for − LIF2d . For Oct4 - EGFP , N = 22 ( high ) , 18 ( middle ) and 17 ( low ) for + 2i , 17 ( high ) 16 ( middle ) and 20 ( low ) for + LIF , 19 ( high ) , 19 ( middle ) and 20 cells ( low ) for − LIF , and 16 ( high ) , 29 ( middle ) and 16 cells ( low ) for − LIF2d . Asterisks and double asterisks indicate p - values of less than 0 . 05 and 0 . 01 , respectively , in Student ' s t - test . Exclamation marks and double exclamation marks indicate p - values of less than 0 . 05 and 0 . 01 , respectively , in Mann Whitney ' s U - test . Note : the data obtained in − LIF2d could not simply compare with the others because the expression levels differed between them .

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

(C, D) Plots of all data of cells categorised with expression level (red, magenta, and blue) and with the mean values (green or cyan) in each parameter in each condition of Nanog-EGFP (C) or Oct4-EGFP (D). The definition of low (blue), middle (magenta), and high (red) in +2i, +LIF, and −LIF were the same as that of Fig. 3F, and those in −LIF2d were the same as B in this figure. Error bars represent standard deviations. For Nanog-EGFP, N = 20 (high), 32 (middle) and 20 (low) for +2i, 26 (high), 18 (middle) and 21 (low) for +LIF, 20 (high), 26 (middle) and 20 cells (low) for −LIF, and 20 (high), 20 (middle) and 19 cells (low) for −LIF2d. For Oct4-EGFP, N = 22 (high), 18 (middle) and 17 (low) for +2i, 17 (high) 16 (middle) and 20 (low) for +LIF, 19 (high), 19 (middle) and 20 cells (low) for −LIF, and 16 (high), 29 (middle) and 16 cells (low) for −LIF2d. Asterisks and double asterisks indicate p-values of less than 0.05 and 0.01, respectively, in Student's t-test. Exclamation marks and double exclamation marks indicate p-values of less than 0.05 and 0.01, respectively, in Mann Whitney's U-test. Note: the data obtained in −LIF2d could not simply compare with the others because the expression levels differed between them.


( A ) Correlation plots of log10g0 and koff of Nanog - EGFP ( left ) and Oct4 - EGFP ( centre ) and log10ξ and koff of Oct4 - EGFP ( right ) in 2i ( top ) , + LIF ( middle ) and − LIF ( bottom ) conditions . Each plot indicates the average value in a single cell . Lines are fitting results with a liner function .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation plots of log10g0 and koff of Nanog-EGFP (left) and Oct4-EGFP (centre) and log10ξ and koff of Oct4-EGFP (right) in 2i (top), +LIF (middle) and −LIF (bottom) conditions. Each plot indicates the average value in a single cell. Lines are fitting results with a liner function.


( D ) Effect of 0 . 5 µM TSA addition on log10g0 of Nanog - EGFP ( left ) and Oct4 - EGFP ( right ) in + LIF ( filled ) and - LIF ( opened ) . Error bars represent standard deviations . For Nanog - EGFP , N = 65 for + LIF - TSA , 91 for + LIF + TSA , 66 for - LIF - TSA , and 88 cells for - LIF + TSA . For Oct4 - EGFP , N = 53 for + LIF - TSA , 42 for + LIF + TSA , 58 for - LIF - TSA , and 39 cells for - LIF + TSA . Exclamation marks and double exclamation marks indicate p - values of less than 0 . 05 and 0 . 01 , respectively , in Mann Whitney ' s U - test .

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

(D) Effect of 0.5 µM TSA addition on log10g0 of Nanog-EGFP (left) and Oct4-EGFP (right) in +LIF (filled) and -LIF (opened). Error bars represent standard deviations. For Nanog-EGFP, N = 65 for +LIF -TSA, 91 for +LIF +TSA, 66 for -LIF -TSA, and 88 cells for -LIF +TSA. For Oct4-EGFP, N = 53 for +LIF -TSA, 42 for +LIF +TSA, 58 for -LIF -TSA, and 39 cells for -LIF +TSA. Exclamation marks and double exclamation marks indicate p-values of less than 0.05 and 0.01, respectively, in Mann Whitney's U-test.


B ) Box - whisker plots of log2 - transformed peptide intensities across all 60 raw files . The color coding highlights the samples plate of origin .

O O O O 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) Box-whisker plots of log2-transformed peptide intensities across all 60 raw files. The color coding highlights the samples plate of origin.


C ) Cumulative frequency curve [ % ] of the observed coefficient of variation ( CV ) of proteins that were identified and quantified with a minimum of three valid values within each day . Here , the ten raw files of each day are evaluated individually . The resulting median and average CV for each day are shown .

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

C) Cumulative frequency curve [%] of the observed coefficient of variation (CV) of proteins that were identified and quantified with a minimum of three valid values within each day. Here, the ten raw files of each day are evaluated individually. The resulting median and average CV for each day are shown.


D ) Pearson correlation heatmap of all 60 raw files and additional sixteen manually prepared HeLa SP3 samples . The displayed data are filtered for 75 % data completeness ( Table 1 ) . Please note the narrow scaling ( 1 - 0 . 94 ) .

O O O O O O O O O O O O O O O O O O O O O O O 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) Pearson correlation heatmap of all 60 raw files and additional sixteen manually prepared HeLa SP3 samples. The displayed data are filtered for 75% data completeness (Table 1). Please note the narrow scaling (1-0.94).


C ) Volcano plot showing differential expression analysis using Limma moderated t - statistics for the comparison of lepidic samples against all other samples . Proteins passing significance thresholds of - log10 p - value < 0 . 05 ( Benjamini - hochberg adjusted ) and an absolute log2 fold change of > 1 are highlighted in orange .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot showing differential expression analysis using Limma moderated t-statistics for the comparison of lepidic samples against all other samples. Proteins passing significance thresholds of -log10 p-value < 0.05 (Benjamini-hochberg adjusted) and an absolute log2 fold change of >1 are highlighted in orange.


B ) Correlation between mg wet tissue weight and the amount of protein extracted by ultrasonication .

B) Correlation between mg wet tissue weight and the amount of protein extracted by ultrasonication.


C ) Pearson correlation heatmap of peptide intensities across five replicates of each sample type ( heart , liver , kidney , HeLa cells ) with the corresponding average .

O O O O O O 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) Pearson correlation heatmap of peptide intensities across five replicates of each sample type (heart, liver, kidney, HeLa cells) with the corresponding average.


D ) The relative number of identified and quantified proteins across the five replicates of each sample type . The average number of identified proteins and the standard deviation across five replicates is shown on top .

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D) The relative number of identified and quantified proteins across the five replicates of each sample type. The average number of identified proteins and the standard deviation across five replicates is shown on top.


E ) Cumulative frequency curve [ % ] of the observed coefficient of variation ( CV ) of proteins that were identified and quantified in at least three out of five replicates in each sample type . The resulting median and average CV for each sample type are shown .

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E) Cumulative frequency curve [%] of the observed coefficient of variation (CV) of proteins that were identified and quantified in at least three out of five replicates in each sample type. The resulting median and average CV for each sample type are shown.


A . Bclaf1 wild type ( WT ) and two Bclaf1 knockout HeLa cell lines ( KO - 1 and - 2 ) were pre - treated with DMSO or cycloheximide ( CHX , 1 μg / ml ) for 30 min , and then treated with TNF ( 10 ng / ml ) together with CHX for 12 hours . Afterwards , the cells were subjected to Annexin V / 7AAD staining followed by flow cytometry analysis , all Annexin V positive cells were counted for analysis , or protein extractions followed by western blotting analysis . B , C . HeLa ( B ) or primary MEFs ( C ) were transfected with control ( siCtrl ) or two siRNAs against Bclaf1 ( siBclaf1 - 1 and - 2 ) and then treated and analyzed as described in A . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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

A. Bclaf1 wild type (WT) and two Bclaf1 knockout HeLa cell lines (KO-1 and -2) were pre-treated with DMSO or cycloheximide (CHX, 1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) together with CHX for 12 hours. Afterwards, the cells were subjected to Annexin V/7AAD staining followed by flow cytometry analysis, all Annexin V positive cells were counted for analysis, or protein extractions followed by western blotting analysis. B, C. HeLa (B) or primary MEFs (C) were transfected with control (siCtrl) or two siRNAs against Bclaf1 (siBclaf1-1 and -2) and then treated and analyzed as described in A. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test.


D , E . MEFs transfected with control ( siCtrl ) or siBclaf1 - 1 and / or siBclaf1 - 2 siRNAs were pre - treated with CHX ( 1 μg / ml ) / z - VAD ( 20 μM ) ( D ) or SM - 164 ( 100 nM ) / z - VAD ( 20 μM ) ( E ) for 30 min , and then added TNF ( 10 ng / ml ) for indicated times . The cells were subjected to Annexin V / 7AAD staining after 9 hours followed by flow cytometry analysis for 7AAD positive and Annexin V negative cells ( up ) , or protein extractions and western blotting analysis at indicated time periods ( down ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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

D, E. MEFs transfected with control (siCtrl) or siBclaf1-1 and/or siBclaf1-2 siRNAs were pre-treated with CHX (1 μg/ml)/z-VAD (20 μM) (D) or SM-164 (100 nM)/z-VAD (20 μM) (E) for 30 min, and then added TNF (10 ng/ml) for indicated times. The cells were subjected to Annexin V/7AAD staining after 9 hours followed by flow cytometry analysis for 7AAD positive and Annexin V negative cells (up), or protein extractions and western blotting analysis at indicated time periods (down). Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test.


A , B . Cell lysates of HeLa Bclaf1 WT and KO cells ( A ) , and HeLa cells transfected with siCtrl or siBclaf1 - 1 ( B ) treated with TNF ( 10 ng / ml ) for indicated time periods were analyzed by western blotting with the indicated antibodies .

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A, B. Cell lysates of HeLa Bclaf1 WT and KO cells (A), and HeLa cells transfected with siCtrl or siBclaf1-1 (B) treated with TNF (10 ng/ml) for indicated time periods were analyzed by western blotting with the indicated antibodies.


C , D . HeLa Bclaf1 WT and KO cells ( C ) , and HeLa cells transfected with siCtrl or siBclaf1 - 1 ( D ) were treated with TNF ( 10 ng / ml ) for the indicated time periods , and then subject to nucleus - cytoplasm fractionation as described in materials and methods followed by western blotting . Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions , respectively .

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C, D. HeLa Bclaf1 WT and KO cells (C), and HeLa cells transfected with siCtrl or siBclaf1-1 (D) were treated with TNF (10 ng/ml) for the indicated time periods, and then subject to nucleus-cytoplasm fractionation as described in materials and methods followed by western blotting. Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively.


B . MEFs treated with TNF ( 10 ng / ml ) for 4 hours were subject to FADD immunoprecipitation , and then analyzed by western blotting .

B. MEFs treated with TNF (10 ng/ml) for 4 hours were subject to FADD immunoprecipitation, and then analyzed by western blotting.


G , H . HeLa cells transfected with siRNAs against Bclaf1 or c - FLIP were pretreated with CHX ( 1 μg / ml ) for 30 min and then stimulated with TNF ( 10 ng / ml ) in the presence of CHX for 12 hours followed by Annexin V / 7AAD staining and flow cytometry analysis ( G ) , or protein extractions followed by western blotting analysis ( H ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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 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

G, H. HeLa cells transfected with siRNAs against Bclaf1 or c-FLIP were pretreated with CHX (1 μg/ml) for 30 min and then stimulated with TNF (10 ng/ml) in the presence of CHX for 12 hours followed by Annexin V/7AAD staining and flow cytometry analysis (G), or protein extractions followed by western blotting analysis (H). Data information: Data are shown as mean ± SD. n=3 biological replicates. **p<0.01; ****p<0.0001. One-way ANOVA test.


A - F . Bclaf1 WT , KO - 1 and - 2 HeLa cells ( A and D ) , as well as HeLa ( B and E ) and primary MEFs ( C and F ) which were transfected with siCtrl or siBclaf1 - 1 and - 2 were treated with TNF ( 10 ng / ml ) for 6 hours followed by western blotting analysis ( A - C ) , or for 4 hours followed by total RNA extraction and RT - PCR analysis ( D - F ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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A-F. Bclaf1 WT, KO-1 and -2 HeLa cells (A and D), as well as HeLa (B and E) and primary MEFs (C and F) which were transfected with siCtrl or siBclaf1-1 and -2 were treated with TNF (10 ng/ml) for 6 hours followed by western blotting analysis (A-C), or for 4 hours followed by total RNA extraction and RT-PCR analysis (D-F). Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test.


H , I . CHIP analysis of the binding of Bclaf1 to the promoter of CFLAR was performed in HeLa - Flag - Bclaf1 cells treated without ( H ) or with TNF ( I ) . The regions amplified by RT - PCR were delineated . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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H, I. CHIP analysis of the binding of Bclaf1 to the promoter of CFLAR was performed in HeLa-Flag-Bclaf1 cells treated without (H) or with TNF (I). The regions amplified by RT-PCR were delineated. Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test.


B . CFLAR promoter were co - transfected with Bclaf1 or / and p50 into HeLa - Bclaf1 - KO cells , and the luciferase activity was measured as described before .

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B. CFLAR promoter were co-transfected with Bclaf1 or/and p50 into HeLa-Bclaf1-KO cells, and the luciferase activity was measured as described before.


E , HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre - treated with DMSO or CHX ( 1 μg / ml ) for 30 min , and then treated with TNF ( 10 ng / ml ) for 12 hours . Cells were lysed and then analyzed by western

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

E, HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre-treated with DMSO or CHX (1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) for 12 hours. Cells were lysed and then analyzed by western blotting


G . CHIP analysis of the CFLAR promoter in HeLa - Flag - Bclaf1 cells . Immunoprecipitation was performed using anti - Flag and control IgG antibodies followed by RT - PCR analysis . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way or Two - way ANOVA test .

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G. CHIP analysis of the CFLAR promoter in HeLa-Flag-Bclaf1 cells. Immunoprecipitation was performed using anti-Flag and control IgG antibodies followed by RT-PCR analysis. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. One-way or Two-way ANOVA test.


A . HeLa - Flag - Bclaf1 and Bclaf1 WT HeLa cells treated with or without TNF ( 10 ng / ml ) for 2 hours were separated into the cytoplasmic and the nuclear fraction . Endogenous Flag - Bclaf1 complex in each fraction was immunoprecipitated with M2 beads and analyzed by western blotting .

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A. HeLa-Flag-Bclaf1 and Bclaf1 WT HeLa cells treated with or without TNF (10 ng/ml) for 2 hours were separated into the cytoplasmic and the nuclear fraction. Endogenous Flag-Bclaf1 complex in each fraction was immunoprecipitated with M2 beads and analyzed by western blotting.


B . HeLa - Flag - Bclaf1 cells transfected with siCtrl or sip65 were treated with TNF ( 10 ng / ml ) for 2 hours . The nuclear fraction was isolated and subjected to immunoprecipitation with an anti - Flag or control IgG antibody , followed by western blotting analysis .

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B. HeLa-Flag-Bclaf1 cells transfected with siCtrl or sip65 were treated with TNF (10 ng/ml) for 2 hours. The nuclear fraction was isolated and subjected to immunoprecipitation with an anti-Flag or control IgG antibody, followed by western blotting analysis.


D . CFLAR FL promoter were co - transfected with p50 and Bclaf1 FL or its individual deletion mutant into HeLa - Bclaf1 - KO cells . The experiments were performed in triplicate with a Renilla reporter in the transfection mixture for normalization . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant . One - way ANOVA test .

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D. CFLAR FL promoter were co-transfected with p50 and Bclaf1 FL or its individual deletion mutant into HeLa-Bclaf1-KO cells. The experiments were performed in triplicate with a Renilla reporter in the transfection mixture for normalization. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant. One-way ANOVA test.


siRNAs against Bclaf1 and control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . The mice were then treated with mTNF for two hours before sacrifice . The small intestines were excised and processed for immunohistochemical staining for Bclaf1 ( B ) and Hematoxylin - and - eosin staining ( C ) Bclaf1 knockdown efficiency is detected by immunohistochemical analysis ( B ) . Hematoxylin - and - eosin staining showing the morphological change ( C ) . Villus height and crypt depth were measured ( D ) . Data are shown as mean ± SD . n = 5 mice for each group . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test . Scale bars : 50 μm in B , 100 μm in C .

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siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for Bclaf1 (B) and Hematoxylin-and-eosin staining (C) Bclaf1 knockdown efficiency is detected by immunohistochemical analysis (B). Hematoxylin-and-eosin staining showing the morphological change (C). Villus height and crypt depth were measured (D). Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μm in B, 100 μm in C.


siRNAs against Bclaf1 and control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . The mice were then treated with mTNF for two hours before sacrifice . The small intestines were excised and processed for immunohistochemical staining for cleaved caspase 3 ( E ) , phospholated RIPK3 ( F ) CC3 + and p - RIPK3 + positive cells in five fields per intestine were quantified . Data are shown as mean ± SD . n = 5 mice for each group . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test . Scale bars : 50

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siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for cleaved caspase 3 (E), phospholated RIPK3 (F) CC3+ and p-RIPK3+ positive cells in five fields per intestine were quantified. Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μm


siRNAs against Bclaf1 and control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . The mice were then treated with mTNF for two hours before sacrifice . The small intestines were excised and processed for western blotting analysis ( G ) .

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siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for western blotting analysis (G).


HIEC - 6 cells were transfected siCtrl or siBclaf1 - 1 and - 2 . Then the cells were treated with TNF for 24 hours were subjected for protein extractions and western blotting analysis ( I and J )

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HIEC-6 cells were transfected siCtrl or siBclaf1-1 and -2. Then the cells were treated with TNF for 24 hours were subjected for protein extractions and western blotting analysis (I and J)


A Electron micrograph of unstained UMOD filament sheets in human urine . The inset highlights a double helix - like structure resulting from juxtaposition of two individual filaments . Scale bars : 50 nm and 10 nm ( inset ) . B , C " Tree " front view and " zig - zag " side view of purified native UMOD filaments , imaged using a Volta phase plate . Scale bars : 10 nm . D , E Front and side views of UMODe filaments , showing absence of branches . Scale bars : 10 nm .

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A Electron micrograph of unstained UMOD filament sheets in human urine. The inset highlights a double helix-like structure resulting from juxtaposition of two individual filaments. Scale bars: 50 nm and 10 nm (inset). B, C "Tree" front view and "zig-zag" side view of purified native UMOD filaments, imaged using a Volta phase plate. Scale bars: 10 nm. D, E Front and side views of UMODe filaments, showing absence of branches. Scale bars: 10 nm.


A Electron micrograph of unstained UMOD filament sheets in human urine . The inset highlights a double helix - like structure resulting from juxtaposition of two individual filaments . Scale bars : 50 nm and 10 nm ( inset ) .

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A Electron micrograph of unstained UMOD filament sheets in human urine. The inset highlights a double helix-like structure resulting from juxtaposition of two individual filaments. Scale bars: 50 nm and 10 nm (inset).


A Immunofluorescence of unpermeabilized stably transfected MDCK cells shows that , unlike wt full - length UMOD , mutants ZP - N ( R415A ) or ZP - C ( ΔFA ) do not assemble into filaments . Scale bar : 50 µm .

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A Immunofluorescence of unpermeabilized stably transfected MDCK cells shows that, unlike wt full-length UMOD, mutants ZP-N (R415A) or ZP-C (ΔFA) do not assemble into filaments. Scale bar: 50 µm.


B - G Immunofluorescence of unpermeabilized cells co - expressing FLAG - tagged wt UMOD ( green ) and the indicated HA - tagged isoforms ( red ) . UMOD R415A does not incorporate into polymers that only contain wt protein ( C ) . UMOD ΔFA has a dominant negative effect on wt protein polymerization ( D ) that is rescued in a double mutant carrying both ZP - N and ZP - C mutations ( F ) . Similarly , the dominant negative effect of a CCS mutation ( 4A ) that prevents EHP dissociation ( E ) is suppressed by the introducing the R415A mutation in the 4A isoform ( G ) . Scale bar : 50 µm .

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B-G Immunofluorescence of unpermeabilized cells co-expressing FLAG-tagged wt UMOD (green) and the indicated HA-tagged isoforms (red). UMOD R415A does not incorporate into polymers that only contain wt protein (C). UMOD ΔFA has a dominant negative effect on wt protein polymerization (D) that is rescued in a double mutant carrying both ZP-N and ZP-C mutations (F). Similarly, the dominant negative effect of a CCS mutation (4A) that prevents EHP dissociation (E) is suppressed by the introducing the R415A mutation in the 4A isoform (G). Scale bar: 50 µm.


H Immunofluorescence of permeabilized cells expressing soluble isoforms of UMOD truncated before the EHP ( UMOD - CCS ) shows that wt forms intracellular polymers whereas polymerization interface mutants do not . The intracellular polymers are localized in the endoplasmic reticulum ( ER ) , as shown by co - staining with the KDEL sequence used as ER marker . Scale bar : 10 µm .

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H Immunofluorescence of permeabilized cells expressing soluble isoforms of UMOD truncated before the EHP (UMOD-CCS) shows that wt forms intracellular polymers whereas polymerization interface mutants do not. The intracellular polymers are localized in the endoplasmic reticulum (ER), as shown by co-staining with the KDEL sequence used as ER marker. Scale bar: 10 µm.


A Analytical SEC of UFE HCE / LCE - digests produces two protein peaks , F1 and F2 .

A Analytical SEC of UFE HCE/LCE-digests produces two protein peaks, F1 and F2.


Reducing SDS - PAGE of SEC purified UFE digests with indicated molecular weight markers . The different SEC elution volumes of F1 and F2 ( A ) reflect different levels of egg coat filament digestion by HCE / LCE .

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Reducing SDS-PAGE of SEC purified UFE digests with indicated molecular weight markers. The different SEC elution volumes of F1 and F2 (A) reflect different levels of egg coat filament digestion by HCE/LCE.


native - PAGE of SEC purified UFE digests with indicated molecular weight markers . The different SEC elution volumes of F1 and F2 ( A ) reflect different levels of egg coat filament digestion by HCE / LCE .

native-PAGE of SEC purified UFE digests with indicated molecular weight markers. The different SEC elution volumes of F1 and F2 (A) reflect different levels of egg coat filament digestion by HCE/LCE.


D , E TOF - MS spectrograms of purified F2 ( D ) and F1 ( E ) products . Round - shaped symbols near the peaks indicate the two moieties of the intact ZP module of ZI - 3 ( green ) or the separated ZP - N and ZP - C domains of LCE - cleaved ZI - 1 , 2 ( purple ) .

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D, E TOF-MS spectrograms of purified F2 (D) and F1 (E) products. Round-shaped symbols near the peaks indicate the two moieties of the intact ZP module of ZI-3 (green) or the separated ZP-N and ZP-C domains of LCE-cleaved ZI-1,2 (purple).


F , G TOF - MS spectrograms of cross - linked products of F2 ( F ) and F1 ( G ) , with domain symbols indicating the deduced subunit composition of cross - linked products .

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F, G TOF-MS spectrograms of cross-linked products of F2 (F) and F1 (G), with domain symbols indicating the deduced subunit composition of cross-linked products.


I Electron micrograph of negatively - stained fish egg coat filaments . On the right are representative 2D classes , indicating a separation of ~ 65 Å between repeating units within the polymers ( white arrows ) . Scale bars : 100 nm ( left panel ) and 10 nm ( bottom right panel ) .

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I Electron micrograph of negatively-stained fish egg coat filaments. On the right are representative 2D classes, indicating a separation of ~65 Å between repeating units within the polymers (white arrows). Scale bars: 100 nm (left panel) and 10 nm (bottom right panel).


A Negative stain micrograph of a sheet formed by purified UMOD filaments in 100 mM NaCl ( top ) . Analysis of this material by correlation averaging reveals a periodic pattern ( bottom ) with a two - fold symmetry axis perpendicular to the plane of the sheet ( white symbol ) . Scale bars : 250 Å ( top ) and 125 Å ( bottom ) .

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A Negative stain micrograph of a sheet formed by purified UMOD filaments in 100 mM NaCl (top). Analysis of this material by correlation averaging reveals a periodic pattern (bottom) with a two-fold symmetry axis perpendicular to the plane of the sheet (white symbol). Scale bars: 250 Å (top) and 125 Å (bottom).


B . Endogenous CUL4A and CUL4B complexes were isolated from 293T cell lysates by immunoprecipitation ( IP ) and associated proteins were detected by immunoblotting ( IB ) . Mock includes beads only control .

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B. Endogenous CUL4A and CUL4B complexes were isolated from 293T cell lysates by immunoprecipitation (IP) and associated proteins were detected by immunoblotting (IB). Mock includes beads only control.


C . Flag - tagged WT and mutant WDTC1proteins were transiently expressed in 293T cells . Flag - WDTC1 complexes were immunoprecipitated with anti - FLAG and their associated proteins were detected by immunoblotting as indicated ; EV , empty vector control .

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

C. Flag-tagged WT and mutant WDTC1proteins were transiently expressed in 293T cells. Flag-WDTC1 complexes were immunoprecipitated with anti-FLAG and their associated proteins were detected by immunoblotting as indicated; EV, empty vector control.


E . 293T cells were transfected with HA - ubiquitin along with various combinations of plasmid and siRNA as indicated . Transfected cells were lysed under denaturing conditions to obtain whole cell extracts ( WCE ) and immunoprecipitated with anti - FLAG . WDTC1 ubiquitylation was evaluated by immunoblotting as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 293T cells were transfected with HA-ubiquitin along with various combinations of plasmid and siRNA as indicated. Transfected cells were lysed under denaturing conditions to obtain whole cell extracts (WCE) and immunoprecipitated with anti-FLAG. WDTC1 ubiquitylation was evaluated by immunoblotting as indicated.


F . 293T cells transiently expressing Flag - WDTC1 were transfected with scramble ( scrm ) siRNA or siRNAs against DDB1 , CUL4A and CUL4B . Flag - WDTC1 levels and knockdown efficiency were assessed by immunoblotting as indicated .

F. 293T cells transiently expressing Flag-WDTC1 were transfected with scramble (scrm) siRNA or siRNAs against DDB1, CUL4A and CUL4B. Flag-WDTC1 levels and knockdown efficiency were assessed by immunoblotting as indicated.


A . 3T3 - L1preadipocytes were adipogenically induced and lysates were prepared from cells collected at indicated days . Endogenous WDTC1 interaction with CRL4 subunits was evaluated by immunoprecipitation with anti - WDTC1 followed by immunoblot analyses with indicated antibodies . Fatty acid synthase ( FAS ) levels were monitored to evaluate induction .

A. 3T3-L1preadipocytes were adipogenically induced and lysates were prepared from cells collected at indicated days. Endogenous WDTC1 interaction with CRL4 subunits was evaluated by immunoprecipitation with anti-WDTC1 followed by immunoblot analyses with indicated antibodies. Fatty acid synthase (FAS) levels were monitored to evaluate induction.


B - D . 3T3 - L1 stable cells expressing vector control ( EV ) , Flag - tagged WT or mutant WDTC1 proteins were adipogenically induced . Adipogenesis was assessed by Oil Red O staining ( ORO ) ( B ) ; plate ( top ) and microscopic ( bottom ) views , 0 . 1 mm scale bar .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Adipogenesis was assessed by Oil Red O staining (ORO) (B); plate (top) and microscopic (bottom) views, 0.1 mm scale bar.


B - D . 3T3 - L1 stable cells expressing vector control ( EV ) , Flag - tagged WT or mutant WDTC1 proteins were adipogenically induced . Triglyceride levels were quantified by an enzymatic assay ( C ) .

O O O O 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

B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Triglyceride levels were quantified by an enzymatic assay (C).


B - D . 3T3 - L1 stable cells expressing vector control ( EV ) , Flag - tagged WT or mutant WDTC1 proteins were adipogenically induced . Adipogenic gene expression was evaluated by RT - qPCR analysis ( D ) .

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

B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Adipogenic gene expression was evaluated by RT-qPCR analysis (D).


A . Schematic of WDTC1 knockdown - rescue lentiviral vector ( top ) . 3T3 - L1preadipocytes were lentivirally transduced to generate stable cells expressing a non - specific ( NS ) shRNA control , shWDTC1 targeting endogenous Wdtc1mRNA , or cells simultaneously expressing shWDTC1 and shRNA - resistant WDTC1 ; bottom , confirmation of knockdown and WDTC1 ectopic expression .

A. Schematic of WDTC1 knockdown-rescue lentiviral vector (top). 3T3-L1preadipocytes were lentivirally transduced to generate stable cells expressing a non-specific (NS) shRNA control, shWDTC1 targeting endogenous Wdtc1mRNA, or cells simultaneously expressing shWDTC1 and shRNA-resistant WDTC1; bottom, confirmation of knockdown and WDTC1 ectopic expression.


B - D . 3T3 - L1 stable cells described in ( A ) were adipogenically induced . Their adipogenic potential was assessed by ORO ( B )

O O O O 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-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by ORO (B)


B - D . 3T3 - L1 stable cells described in ( A ) were adipogenically induced . Their adipogenic potential was assessed by triglyceride quantitation ( C )

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

B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by triglyceride quantitation (C)


B - D . 3T3 - L1 stable cells described in ( A ) were adipogenically induced . Their adipogenic potential was assessed by adipogenic gene expression ( D ) .

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

B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by adipogenic gene expression (D).


A . Induced 3T3 - L1 cells stably expressing Flag - WDTC1 were subjected to subcellular fractionation . Aliquots of total cell extract ( TCE ) and subcellular fractions were analyzed by immunoblot as indicated ; fraction purity was assessed by α - Tubulin and histone H3 antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O 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. Induced 3T3-L1 cells stably expressing Flag-WDTC1 were subjected to subcellular fractionation. Aliquots of total cell extract (TCE) and subcellular fractions were analyzed by immunoblot as indicated; fraction purity was assessed by α-Tubulin and histone H3 antibodies.


B . Chromatin enriched nuclear extracts from 293T cells cotransfected with Flag - WDTC1 and HA tagged core histones were immunoprecipitated ( IP ) with anti - FLAG and immunoblotted ( IB ) as indicated . The probable H2AK119ub1 band detected in the nuclear extract is indicated by an asterisk .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Chromatin enriched nuclear extracts from 293T cells cotransfected with Flag-WDTC1 and HA tagged core histones were immunoprecipitated (IP) with anti-FLAG and immunoblotted (IB) as indicated. The probable H2AK119ub1 band detected in the nuclear extract is indicated by an asterisk.


C . Soluble chromatin extracts were prepared from preadipocyte or induced 3T3 - L1 cells stably expressing control ( EV ) and Flag - WDTC1 proteins . H2A monoubiquitylation was detected by H2AK119ub1 - specific antibody and total H2A served as loading control . Relative intensity represents ratio of H2AK119ub1 over total H2A signal normalized to EV control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Soluble chromatin extracts were prepared from preadipocyte or induced 3T3-L1 cells stably expressing control (EV) and Flag-WDTC1 proteins. H2A monoubiquitylation was detected by H2AK119ub1-specific antibody and total H2A served as loading control. Relative intensity represents ratio of H2AK119ub1 over total H2A signal normalized to EV control.


D . Affinity purified Flag - WDTC1 wildtype and mutant ΔH complexes from 293T cells were the source of E3 ligase in in vitro ubiquitylation assays . Copurification of CRL4 E3 ligase proteins was confirmed by immunoblotting and Coomassie Blue staining ( left panel ) . In vitro ubiquitylation assays of recombinant H2A were performed in the presence of indicated proteins . Reaction products were resolved by SDS - PAGE , and ubiquitylatedH2A was detected by anti - H2AK119ub1 ( right panel ) .

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

D. Affinity purified Flag-WDTC1 wildtype and mutant ΔH complexes from 293T cells were the source of E3 ligase in in vitro ubiquitylation assays. Copurification of CRL4 E3 ligase proteins was confirmed by immunoblotting and Coomassie Blue staining (left panel). In vitro ubiquitylation assays of recombinant H2A were performed in the presence of indicated proteins. Reaction products were resolved by SDS-PAGE, and ubiquitylatedH2A was detected by anti-H2AK119ub1 (right panel).


E . In vitro ubiquitylation assays were performed with purified wild - type and mutant histone proteins as substrates . H2A ubiquitylation was assessed by immunoblotting with anti - ubiquitin and various antibodies as indicated .

O O O O O O O O O O O O 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. In vitro ubiquitylation assays were performed with purified wild-type and mutant histone proteins as substrates. H2A ubiquitylation was assessed by immunoblotting with anti-ubiquitin and various antibodies as indicated.


A . Four representative images of RHOB immunostaining in human non - small - cell lung cancers . Figures correspond to the percentage of tumor analyzed .

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A. Four representative images of RHOB immunostaining in human non-small-cell lung cancers. Figures correspond to the percentage of tumor analyzed.


B . Characteristics of 96 patients with an EGFR - activating mutation who were treated with EGFR - TKI . Each column corresponds to one patient . Data include general characteristics ( stage at diagnosis , age , sex , tobacco usage , position of EGFR mutation , TKI type ) , progression - free survival ( PFS ; lower graph ) and intensity of RHOB staining as determined by immunohistochemistry ( null : 0 , weak : + , moderate : + + and high : + + + ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Characteristics of 96 patients with an EGFR-activating mutation who were treated with EGFR-TKI. Each column corresponds to one patient. Data include general characteristics (stage at diagnosis, age, sex, tobacco usage, position of EGFR mutation, TKI type), progression-free survival (PFS; lower graph) and intensity of RHOB staining as determined by immunohistochemistry (null: 0, weak: +, moderate: ++ and high: +++).


C . Representative scans of patients with low or high RHOB - expressing lung tumors , before and after erlotinib treatment . Red arrows indicate lung tumors .

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C. Representative scans of patients with low or high RHOB-expressing lung tumors, before and after erlotinib treatment. Red arrows indicate lung tumors.


D . Progression - free survival of erlotinib - treated patients with EGFR - mutated lung tumors , according to RHOB expression , assessed by immunohistochemistry ( low RHOB group = negative + weak staining ; high RHOB group = moderate + high staining ) .

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

D. Progression-free survival of erlotinib-treated patients with EGFR-mutated lung tumors, according to RHOB expression, assessed by immunohistochemistry (low RHOB group = negative + weak staining; high RHOB group = moderate + high staining).


E . Progression - free survival of patients who received EGFR - TKi as first - line therapy ( n = 63 ) . F . Progression - free survival of patients who received EGFR - TKi as 2nd - line ( n = 28 ) , 3rd - line ( n = 3 ) or 4th - line ( n = 2 ) therapy .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Progression-free survival of patients who received EGFR-TKi as first-line therapy (n=63). F. Progression-free survival of patients who received EGFR-TKi as 2nd-line (n=28), 3rd-line (n=3) or 4th-line (n=2) therapy.


G . RHOB immunostaining score evolution in EGFR - mutated lung tumors before treatment and after EGFR - TKI relapse .

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

G. RHOB immunostaining score evolution in EGFR-mutated lung tumors before treatment and after EGFR-TKI relapse.


A . Representative H & amp ; amp ; E staining of whole lungs from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days . Scale bar : 5 mm . B . Quantification of the tumor / lung ratio . n = 7 for each group except for EGFRL858R / Rhob + / - placebo ( n = 6 ) .

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

A. Representative H&amp;E staining of whole lungs from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. Scale bar: 5 mm. B. Quantification of the tumor/lung ratio. n=7 for each group except for EGFRL858R/Rhob+/- placebo (n=6).


C . Representative Ki67 immunostaining of EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days ( scale bar : 50 µm ) , and the corresponding quantification ( D ) . Three independent zones / mice lung were used for quantification . n = 21 ( 7 mice ) for each group except for EGFRL858R / Rhob + / - placebo ( n = 18 ; 6 mice ) .

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

C. Representative Ki67 immunostaining of EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D). Three independent zones/mice lung were used for quantification. n=21 (7 mice) for each group except for EGFRL858R/Rhob+/- placebo (n=18; 6 mice).


E . Immunostaining of cleaved caspase - 3 in lung tumors from EGFRL858R / Rhob + / + or EGFRL858R / Rhob - / - mice treated for 24 h with erlotinib at 12 . 5 mg / kg . Scale bar : 50 µm . ( * * : p < 0 . 001 vs . placebo ; * * * : p < 0 . 0001 vs . placebo ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunostaining of cleaved caspase-3 in lung tumors from EGFRL858R/Rhob+/+ or EGFRL858R/Rhob-/- mice treated for 24 h with erlotinib at 12.5 mg/kg. Scale bar: 50 µm. (**: p<0.001 vs. placebo; ***: p<0.0001 vs. placebo).


HCC4006 cells were ( A ) transfected with two siRNA against RHOB ( siB1 , siB2 ) then treated with increasing doses of erlotinib . The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells .

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

HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.


HCC4006 cells were ( A ) transfected with two siRNA against RHOB ( siB1 , siB2 ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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

HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


HCC4006 cells were ( B ) transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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

HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


HCC4006 cells were ( B ) transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) then treated with increasing doses of erlotinib . The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.


HCC4006 cells were ( C ) both transfected with siRNA and transduced by adenoviruses ; then treated with increasing doses of erlotinib . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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

HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


HCC4006 cells were ( C ) both transfected with siRNA and transduced by adenoviruses ; then treated with increasing doses of erlotinib . The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells .

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

HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.


Erlotinib IC50 values were quantified in RHOB - overexpressing or RHOB - depleted ( D ) HCC827 cells , ( E ) HCC2935 cells and ( F ) H3255 cells , as determined by an MTS assay after 72 h treatment . RHOB overexpression or inhibition was monitored by western blotting for each condition .

Erlotinib IC50 values were quantified in RHOB-overexpressing or RHOB-depleted (D) HCC827 cells, (E) HCC2935 cells and (F) H3255 cells, as determined by an MTS assay after 72 h treatment. RHOB overexpression or inhibition was monitored by western blotting for each condition.


G . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) at an increasing multiplicity of infection ( MOI ) , then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed .

O O O O O O 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

G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI), then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed.


G . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) at an increasing multiplicity of infection ( MOI ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control cells ) . Data are representative of at least three independent experiments .

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

G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


A . HCC4006 , HCC827 , HCC2935 and H3255 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for four hours with erlotinib at concentrations corresponding to the respective IC50 values determined for each control cell line . The phosphorylation status of AKT , ERK1 / 2 and EGFR was assessed by western blotting and normalized according to total protein levels . RHOB overexpression was also monitored 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 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

A. HCC4006, HCC827, HCC2935 and H3255 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib at concentrations corresponding to the respective IC50 values determined for each control cell line. The phosphorylation status of AKT, ERK1/2 and EGFR was assessed by western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by western blotting.


B . Representative immunostaining of phospho - AKT ( Ser473 ) , phospho ERK1 / 2 and their total protein amounts in lung tumors from EGFRL858R / Rhob - / - or EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days . The remaining hyperplastic areas were selected in erlotinib - treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes . 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

B. Representative immunostaining of phospho-AKT (Ser473), phospho ERK1/2 and their total protein amounts in lung tumors from EGFRL858R/Rhob-/- or EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. The remaining hyperplastic areas were selected in erlotinib-treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bar: 100 µm.


C . HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant ( AKTmyr , myristoylated ) or an empty vector ( ø ) , and treated for 72 h with increasing concentrations of erlotinib . The surviving cell fraction was determined by an MTS assay . Data are representative of at least three independent experiments .

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

C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay. Data are representative of at least three independent experiments.


C . HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant ( AKTmyr , myristoylated ) or an empty vector ( ø ) , and treated for 72 h with increasing concentrations of erlotinib . AKT overexpression and phosphorylation at Ser473 were assessed by western blotting . Data are representative of at least three independent experiments .

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

C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. AKT overexpression and phosphorylation at Ser473 were assessed by western blotting. Data are representative of at least three independent experiments.


A . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for four hours with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . The phosphorylation status of GSK3β ( Ser9 ) , ERK1 / 2 and EGFR ( Tyr1173 ) was assessed by western blotting and normalized according to the total protein levels . RHOB overexpression was also monitored 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2 and EGFR (Tyr1173) was assessed by western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by western blotting.


B . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 72 h with erlotinib alone ( black and red curves ) or in combination with the AKT inhibitor G594 at 100 nM ( green and blue curves ) . The surviving cell fraction was determined by an MTS assay .

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

B. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 72 h with erlotinib alone (black and red curves) or in combination with the AKT inhibitor G594 at 100 nM (green and blue curves). The surviving cell fraction was determined by an MTS assay.


C . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594 . The surviving cell fraction was determined by an MTS assay , and erlotinib IC50 values were determined for each condition . ( * * : p < 0 . 001 vs . AdCont cells ; * * * : p < 0 . 0001 vs . AdCont cells ) . HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs .

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C. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594. The surviving cell fraction was determined by an MTS assay, and erlotinib IC50 values were determined for each condition. (**: p<0.001 vs. AdCont cells; ***: p<0.0001 vs. AdCont cells). HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs.


HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . Apoptosis was then determined by quantification of the subG1 cell population ( D ) .

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

HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by quantification of the subG1 cell population (D).


HCC4006 cells were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses and treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . Apoptosis was then determined by detection of cleaved PARP and caspase - 3 ( E ) .

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

HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by detection of cleaved PARP and caspase-3 (E).


A . Representative H & amp ; amp ; E staining of lung tumors from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not during four days with erlotinib ( 12 . 5 mg / kg / day ) , with the AKT inhibitor G594 ( 25 mg / kg / day ) , or in combination with both drugs . Scale bar : 500 µm . B . Quantification of the tumor / lung ratio of mice treated or not with the individual drugs or with a combination of both .

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

A. Representative H&amp;E staining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not during four days with erlotinib (12.5 mg/kg/day), with the AKT inhibitor G594 (25 mg/kg/day), or in combination with both drugs. Scale bar: 500 µm. B. Quantification of the tumor/lung ratio of mice treated or not with the individual drugs or with a combination of both.


C . Representative Ki67 immunostaining of lung tumors from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - and EGFRL858R / Rhob + / + mice treated or not with erlotinib alone ( 12 . 5 mg / kg / day ) or in combination with the AKT inhibitor G594 ( 25 mg / kg / day ) for four days ( scale bar : 50 µm ) , and the corresponding quantification ( D ) .

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

C. Representative Ki67 immunostaining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib alone (12.5 mg/kg/day) or in combination with the AKT inhibitor G594 (25 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D).


A ) Distribution curve of replication fork rates for Mybl2 + / + and Mybl2Δ / Δ ESCs . Statistical analysis was performed using the Mann Whitney U test . n = 6 experimental replicates . A minimum of 490 replication forks were counted . ( * * * * p < 0 . 0001 ) .

A) Distribution curve of replication fork rates for Mybl2+/+ and Mybl2Δ/Δ ESCs. Statistical analysis was performed using the Mann Whitney U test. n=6 experimental replicates. A minimum of 490 replication forks were counted.( **** p< 0.0001).


B ) Left panel : representative elongating replication forks ( stable and unstable ) . Quantification of the average IdU / CldU ratio in the Mybl2 + / + and Mybl2Δ / Δ ESCs . Percentage of highly unstable forks ( ratio above 2 ) are indicated . Y - axis was cut to change scale and display large values . Statistical analysis was performed using the Mann Whitney U test . ( * * * * p < 0 . 0001 ) . At least 450 ratios were recorded from 6 experimental 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: representative elongating replication forks (stable and unstable). Quantification of the average IdU/CldU ratio in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Percentage of highly unstable forks (ratio above 2) are indicated. Y-axis was cut to change scale and display large values. Statistical analysis was performed using the Mann Whitney U test. (**** p< 0.0001). At least 450 ratios were recorded from 6 experimental replicates.


C ) The length of both CldU labels surrounding IdU tracks ( newly fired origins ) was measured before calculating a ratio representing symmetry around new origins . Quantification of the average positive ratio in the Mybl2 + / + and Mybl2Δ / Δ ESCs . Y - axis was cut to change scale and display large values . Statistical analysis was performed using the Mann Whitney U test . 55 and 75 ratios were recorded in the Mybl2 + / + and Mybl2Δ / Δ respectively from 3 replicates ( * * * * 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 O O 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) The length of both CldU labels surrounding IdU tracks (newly fired origins) was measured before calculating a ratio representing symmetry around new origins. Quantification of the average positive ratio in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Y-axis was cut to change scale and display large values. Statistical analysis was performed using the Mann Whitney U test. 55 and 75 ratios were recorded in the Mybl2+/+ and Mybl2Δ/Δ respectively from 3 replicates ( **** p< 0.0001).


D ) Representative image showing the presence of UFB by positive Immunostaining for PICH . ( Scale bar 10μm ) . Frequency of anaphases positive for UFBs relative to all anaphases in the Mybl2 + / + and Mybl2Δ / Δ ESCs . At least 75 anaphases were counted per experimental group from 3 independent experiments . Statistical analysis was performed using an unpaired two - tailed t - test ( * * * * 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 O O O O O O

D) Representative image showing the presence of UFB by positive Immunostaining for PICH. (Scale bar 10μm). Frequency of anaphases positive for UFBs relative to all anaphases in the Mybl2+/+ and Mybl2Δ/Δ ESCs. At least 75 anaphases were counted per experimental group from 3 independent experiments. Statistical analysis was performed using an unpaired two-tailed t-test (**** p< 0.0001).


E ) Representative images for comets in Mybl2 + / + and Mybl2Δ / Δ untreated and Mybl2 + / + and Mybl2Δ / Δ plus CPT ( 20x magnification ) . ( Scale bar 50μm ) .

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E) Representative images for comets in Mybl2+/+ and Mybl2Δ/Δ untreated and Mybl2+/+ and Mybl2Δ/Δ plus CPT (20x magnification). (Scale bar 50μm).


F ) Quantification of the mean and distributions of olive tail moments in Mybl2 + / + and Mybl2Δ / Δ untreated and CPT - treated . Y - axis was cut to change scale and display large values . Statistical analysis using Mann Whitney U tests . At least 300 comets were measured for each group from 5 experimental replicates ( * * * * 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 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

F) Quantification of the mean and distributions of olive tail moments in Mybl2+/+ and Mybl2Δ/Δ untreated and CPT-treated. Y-axis was cut to change scale and display large values. Statistical analysis using Mann Whitney U tests. At least 300 comets were measured for each group from 5 experimental replicates (**** p< 0.0001).


G ) Frequency of EdU positive cells with over 6 53BP1 foci in Mybl2 + / + , Mybl2Δ / Δ untreated and Mybl2 + / + and Mybl2Δ / Δ plus CPT . Error bars represent SEM . Statistical analysis using unpaired two - tailed t - test . At least 200 EdU positive nuclei were analysed from 4 experimental 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 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

G) Frequency of EdU positive cells with over 6 53BP1 foci in Mybl2+/+, Mybl2Δ/Δ untreated and Mybl2+/+ and Mybl2Δ/Δ plus CPT. Error bars represent SEM. Statistical analysis using unpaired two-tailed t-test. At least 200 EdU positive nuclei were analysed from 4 experimental repeats.


A ) Cells were treated for 1 . 5 hours with 5µM CPT , before sequential addition of IdU and CldU for 20 minutes each . Frequency of new firing origins ( relative to total structures counted ) from Mybl2 + / + and Mybl2Δ / Δ ESCs treated or not with CPT ( n = 3 independent experiments ; Error bars indicate SEM ) . Statistical analysis using two - tailed unpaired t - test .

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

A) Cells were treated for 1.5 hours with 5µM CPT, before sequential addition of IdU and CldU for 20 minutes each. Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with CPT (n=3 independent experiments; Error bars indicate SEM). Statistical analysis using two- tailed unpaired t-test.


( B ) Distribution of replication rate in Mybl2 + / + and Mybl2Δ / Δ ESC treated or not with CPT as above . Statistical analysis was carried out using unpaired Mann Whitney U tests . A minimum of 200 forks were quantified for each of the genotypes and conditions shown from 3 independent repeats ( * p < 0 . 05 ; * * 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 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

(B) Distribution of replication rate in Mybl2+/+ and Mybl2Δ/Δ ESC treated or not with CPT as above. Statistical analysis was carried out using unpaired Mann Whitney U tests. A minimum of 200 forks were quantified for each of the genotypes and conditions shown from 3 independent repeats (*p < 0.05; **p< 0.01; ***p< 0.001; **** p< 0.0001).


C ) ESCs were treated with or without CPT for 4 hours before 0 . 1ug / ml colcemid was added during the final 3 . 5 hours of treatment . Representative image of H3 - pS10 positive cells ( turquoise ) . ( Scale bar 10μm ) . Frequency of H3 - pS10 positive cells in Mybl2 + / + and Mybl2Δ / Δ ESCs . Data from three independent experiments . Error bars represent SEM . Statistical analysis was carried out using a two - tailed unpaired t - test ( * p < 0 . 05 ; * * p < 0 . 01 ; ns = no significant ) .

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

C) ESCs were treated with or without CPT for 4 hours before 0.1ug/ml colcemid was added during the final 3.5 hours of treatment. Representative image of H3-pS10 positive cells (turquoise). (Scale bar 10μm). Frequency of H3-pS10 positive cells in Mybl2+/+ and Mybl2Δ/Δ ESCs. Data from three independent experiments. Error bars represent SEM. Statistical analysis was carried out using a two-tailed unpaired t-test (*p < 0.05; **p< 0.01; ns= no significant).


D ) P - CDK1 ( Tyr15 ) , CDK1 and Beta - actin expression levels of Mybl2 + / + and Mybl2Δ / Δ ESCs with or without CPT treatment analyzed by immunoblotting . Bar graph represents average band density of P - CDK1 in Mybl2Δ / Δ ESCs relative to loading control and relative to the MYBL2 + / + from 6 repeats . Error bars represent SEM . Statistical analysis was carried out using a two - tailed unpaired t - test ( * * p < 0 . 01 ) .

O O O O O 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

D) P-CDK1 (Tyr15), CDK1 and Beta-actin expression levels of Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment analyzed by immunoblotting. Bar graph represents average band density of P-CDK1 in Mybl2Δ/Δ ESCs relative to loading control and relative to the MYBL2+/+ from 6 repeats. Error bars represent SEM. Statistical analysis was carried out using a two-tailed unpaired t-test (**p< 0.01).


A ) Immunoblot showing the levels of phosphorylated CHK1 ( Ser345 ) and GAPDH in the Mybl2 + / + and Mybl2Δ / Δ ESCs with or without CPT treatment ( 2 . 5µM CPT for 4 hours ) .

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A) Immunoblot showing the levels of phosphorylated CHK1 (Ser345) and GAPDH in the Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment (2.5µM CPT for 4 hours).


B ) Immunoblot showing the levels of P - CHK1 ( Ser345 ) and CHK1 in irradiated Mybl2 + / + , and Mybl2Δ / Δ ESCs with or without 3 hours inhibitor treatment : ATR ( AZ20 , 5µM ) and ATM ( Ku60019 , 10µM ) . Cells were also exposed to 5Gy irradiation before the final hour of inhibitor treatment . Bar graph ( lower panel ) represents average band density of P - CHK1 in Mybl2Δ / Δ ESCs relative to CHK1 and relative to untreated cells . Mybl2 + / + from two independent repeats and Mybl2Δ / Δ ESCs from three independent repeats . Error bars represent SD . Statistical analysis was carried out using a two - tailed unpaired t - test ( * p < 0 . 05 ) .

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

B) Immunoblot showing the levels of P-CHK1 (Ser345) and CHK1 in irradiated Mybl2+/+, and Mybl2Δ/Δ ESCs with or without 3 hours inhibitor treatment: ATR (AZ20, 5µM) and ATM (Ku60019, 10µM). Cells were also exposed to 5Gy irradiation before the final hour of inhibitor treatment. Bar graph (lower panel) represents average band density of P-CHK1 in Mybl2Δ/Δ ESCs relative to CHK1 and relative to untreated cells. Mybl2+/+ from two independent repeats and Mybl2Δ/Δ ESCs from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test (*p< 0.05).


C ) Cells were treated for 1 . 5 hours with 5µM AZ20 , before sequential addition of IdU and CldU for 20 minutes each . Dot plot representing the effect of ATR inhibition upon replication rate in Mybl2 + / + and Mybl2Δ / Δ ESC . Statistical analysis of distributions was carried out using unpaired Mann Whitney U tests . A minimum of 240 forks were quantified for each of the genotypes and conditions shown from 2 independent repeats ( * * * * p < 0 . 0001 )

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

C) Cells were treated for 1.5 hours with 5µM AZ20, before sequential addition of IdU and CldU for 20 minutes each. Dot plot representing the effect of ATR inhibition upon replication rate in Mybl2+/+ and Mybl2Δ/Δ ESC. Statistical analysis of distributions was carried out using unpaired Mann Whitney U tests. A minimum of 240 forks were quantified for each of the genotypes and conditions shown from 2 independent repeats (**** p< 0.0001)


D ) Frequency of new firing origins ( relative to total structures counted ) from Mybl2 + / + and Mybl2Δ / Δ ESCs treated or not with ATR inhibitor AZ20 . At least 500 replication structures were counted per treatment from two independent repeats . Statistical analysis using two - tailed unpaired t - test . ( * p < 0 . 05 ; ns = no 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 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

D) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with ATR inhibitor AZ20. At least 500 replication structures were counted per treatment from two independent repeats. Statistical analysis using two- tailed unpaired t-test.(*p< 0.05; ns= no significant).


E ) Distribution and average of IdU / CldU fork ratio in Mybl2 + / + and Mybl2Δ / Δ ESCs with or without ATR inhibitor treatment . Percentage of highly unstable forks ( ratio above 2 ) are indicated . Dotted line at 1 indicates positive ratio . Y - axis was cut to change scale and display large values . Statistical analysis was carried out using the Mann Whitney U test . At least 150 ratios were calculated per treatment group from two independent repeats ( * p < 0 . 05 ; * * 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 O O O O O O O O O O O O O O O O O O O O O O O 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) Distribution and average of IdU/CldU fork ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATR inhibitor treatment. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U test. At least 150 ratios were calculated per treatment group from two independent repeats (*p < 0.05; **p< 0.01; ***p< 0.001; **** p< 0.0001).


B ) Distribution of replication rate after treatment with / without ATM inhibitor . Statistical analysis of distributions was carried out using an unpaired Mann Whitney U test . n = 4 . A minimum of 270 forks was measured for each condition ( * * * * p < 0 . 0001 ) .

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B) Distribution of replication rate after treatment with/without ATM inhibitor. Statistical analysis of distributions was carried out using an unpaired Mann Whitney U test. n=4. A minimum of 270 forks was measured for each condition ( **** p< 0.0001).


C ) Distribution of the average IdU / CldU ratio in Mybl2 + / + and Mybl2Δ / Δ ESCs with or without ATM inhibitor treatment . Percentage of highly unstable forks ( ratio above 2 ) are indicated . Dotted line at 1 indicates positive ratio . Y - axis was cut to change scale and display large values . Statistical analysis was carried out using the Mann Whitney U . At least 260 ratios were calculated per treatment group from 4 separate experiments ( * * * * 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 O O O O O O O O O O O O O O O O O O O O O

C) Distribution of the average IdU/CldU ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATM inhibitor treatment. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U. At least 260 ratios were calculated per treatment group from 4 separate experiments ( **** p< 0.0001).