Figure 8A . Western blot analysis to detect phosphorylation levels of Akt ( S473 ) on lysates from Pmp22 - / - and WT mice at P20 , with quantification . Results are mean ± SEM , p = 0 . 6857 , two - tailed Mann Whitney U test , representative of three independent experiments . B . Western blot analysis for Mbp and P0 on lysates from Pmp22 - / - and WT mice P20 shows decreased myelin protein expression levels at both time points in Pmp22 - / - nerves , results are mean ± SEM , p = 0 . 0500 with the exception of the P0 protein level at P20 , for which the difference between Pmp22 - / - and WT nerves is not statistically significant , p = 0 . 1000 , one - tailed Mann Whitney U test , representative of two independent experiments . C . Representative images of semithin section analysis of WT and Pmp22 - / - sciatic nerves at P30 . Tomacula are abundant in Pmp22 - / - nerves at this age , where both fibers with thicker myelin ( in the range of < 3 - 4 μm in diameter ) and thinner myelin ( with diameters greater than 4 μm , particularly in motor fasicles ) can be observed . C ' . Niaspan does not ameliorate the phenotype in Pmp22 - / - mice , as indicated by the number of tomacula ( quantification of the percentage of tomacula in the entire sciatic nerve section , results are mean ± SEM , p = 0 . 7400 , Mann Whitney U test , n = 15 animals per condition ) . D . Growth curve of Pmp22 - / - mice treated with Niaspan administered by i . p . injection daily at 160mg / Kg and starting at P15 for 15 days . A significant time by group effect is noted between WT saline and Pmp22 - / - saline ( group effect p = 0 . 9907 and time effect p = 0 . 0057 ) , indicating that the two groups start with similar weight values but then the WT saline group ( n = 6 animals ) grows more in time . When Pmp22 - / - saline ( n = 5 ) and Pmp22 - / - Niaspan - treated ( n = 6 ) are compared , the group effect is statistically significant ( p = 0 . 0076 ) while the time effect is not ( p = 0 . 6677 ) , suggesting that these two groups have a similar growth trend but Pmp22 - / - Niaspan - treated group starts with a higher baseline weight . E . Niaspan does not ameliorate myelin thickness as indicated by g - ratio analysis , with axonal diameter distribution : WT 0 . 68 ± 0 . 003 , 2945 fibers , n = 5 animals ; Pmp22 - / - 0 . 64 ± 0 . 003 , 1557 fibers , n = 6 animals ; Pmp22 - / - Niaspan 0 . 63 ± 0 . 003 , 1445 fibers , n = 6 animals , results are mean ± SEM . WT saline as compared to Pmp22 - / - saline - treated p < 0 . 0001 ; WT saline as compared to Pmp22 - / - Niaspan - treated p < 0 . 0001 , repeated measures ANOVA . Note the loss of myelinated fibers in Pmp22 - / - nerves for all caliber axons .

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

Figure 8A. Western blot analysis to detect phosphorylation levels of Akt (S473) on lysates from Pmp22-/- and WT mice at P20, with quantification. Results are mean ± SEM, p=0.6857, two-tailed Mann Whitney U test, representative of three independent experiments.B. Western blot analysis for Mbp and P0 on lysates from Pmp22-/- and WT mice P20 shows decreased myelin protein expression levels at both time points in Pmp22-/- nerves, results are mean ± SEM, p=0.0500 with the exception of the P0 protein level at P20, for which the difference between Pmp22-/- and WT nerves is not statistically significant, p=0.1000, one-tailed Mann Whitney U test, representative of two independent experiments.C. Representative images of semithin section analysis of WT and Pmp22-/- sciatic nerves at P30. Tomacula are abundant in Pmp22-/- nerves at this age, where both fibers with thicker myelin (in the range of <3-4 μm in diameter) and thinner myelin (with diameters greater than 4 μm, particularly in motor fasicles) can be observed.C'. Niaspan does not ameliorate the phenotype in Pmp22-/- mice, as indicated by the number of tomacula (quantification of the percentage of tomacula in the entire sciatic nerve section, results are mean ± SEM, p=0.7400, Mann Whitney U test, n= 15 animals per condition).D. Growth curve of Pmp22-/- mice treated with Niaspan administered by i.p. injection daily at 160mg/Kg and starting at P15 for 15 days. A significant time by group effect is noted between WT saline and Pmp22-/- saline (group effect p=0.9907 and time effect p=0.0057), indicating that the two groups start with similar weight values but then the WT saline group (n=6 animals) grows more in time. When Pmp22-/- saline (n=5) and Pmp22-/- Niaspan-treated (n=6) are compared, the group effect is statistically significant (p=0.0076) while the time effect is not (p=0.6677), suggesting that these two groups have a similar growth trend but Pmp22-/- Niaspan-treated group starts with a higher baseline weight.E. Niaspan does not ameliorate myelin thickness as indicated by g-ratio analysis, with axonal diameter distribution: WT 0.68 ± 0.003, 2945 fibers, n=5 animals; Pmp22-/- 0.64 ± 0.003, 1557 fibers, n= 6 animals; Pmp22-/- Niaspan 0.63 ± 0.003, 1445 fibers, n=6 animals, results are mean ± SEM. WT saline as compared to Pmp22-/- saline-treated p<0.0001; WT saline as compared to Pmp22-/- Niaspan-treated p<0.0001, repeated measures ANOVA. Note the loss of myelinated fibers in Pmp22-/- nerves for all caliber axons.


Figure 2 ( L - N ) B16N parental , O1C44 , and O1DN cells were subcutaneously injected in the right flank of syngeneic C57Bl6 mice revealing the presence of O1C44 ( but not B16N - WT ) cells in abdominal organs and within the peritoneal wall ( L ) . ( M ) H & E stains of the peritoneal wall revealing the presence of melanoma cells in the peritoneal wall . ( N ) Quantitative measurement of degree of invasion within and through the peritoneal wall . Data was analyzed using the Kruskal - Wallis Rank Sum test ( P < 0 . 001 ) . Within all panels , differences established through multiple comparisons are labeled as * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

Figure 2(L-N) B16N parental, O1C44, and O1DN cells were subcutaneously injected in the right flank of syngeneic C57Bl6 mice revealing the presence of O1C44 (but not B16N-WT) cells in abdominal organs and within the peritoneal wall (L). (M) H&E stains of the peritoneal wall revealing the presence of melanoma cells in the peritoneal wall. (N) Quantitative measurement of degree of invasion within and through the peritoneal wall. Data was analyzed using the Kruskal-Wallis Rank Sum test (P < 0.001). Within all panels, differences established through multiple comparisons are labeled as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


Figure 3 ( D - F ) Confluent layers of WM983 cells treated as indicated were ' scratched ' followed by a 20 hour incubation at 37 ° C with 5 % CO2 . ( D ) Representative images of cells after being scratched after being incubated for 0 , 10 or 20 hours in the absence ( - ) or presence ( + ) of 2uM IA65 . ( E - F ) The cell - free area was quantified by ImageJ ( N ≥ 8 biological replicates ) ; data displayed are mean ± SEM . Data was analyzed by 2 - way ANOVA . In the absence of IA65 , SOCE suppression effect was p < 0 . 05 ; effect of time was p < 0 . 0001 ; interaction was p < 0 . 05 . In the presence of IA65 , SOCE suppression effect was p > 0 . 05 ; effect of time was p < 0 . 0001 ; interaction was p > 0 . 05 . Within all panels , differences established through multiple comparisons are labeled as * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

Figure 3(D-F) Confluent layers of WM983 cells treated as indicated were 'scratched' followed by a 20 hour incubation at 37°C with 5% CO2. (D) Representative images of cells after being scratched after being incubated for 0, 10 or 20 hours in the absence (-) or presence (+) of 2uM IA65. (E-F) The cell-free area was quantified by ImageJ (N≥8 biological replicates); data displayed are mean±SEM. Data was analyzed by 2-way ANOVA. In the absence of IA65, SOCE suppression effect was p<0.05; effect of time was p < 0.0001; interaction was p<0.05. In the presence of IA65, SOCE suppression effect was p>0.05; effect of time was p < 0.0001; interaction was p>0.05. Within all panels, differences established through multiple comparisons are labeled as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


Figure 4WM983 and UACC257 human melanoma cells were transfected with Perfringolysin O ( PFO ) - mCherry D4H cholesterol sensor and plated on glass coverslips . Cells were exposed to UV ( 0 or 175 J / m2 ) and incubated for 0 , 2 or 5 weeks as indicated . ( H ) Representative cell images . ( I ) Fluorescence intensity from cells analyzed in ( H ) was measured and quantified using LASX analysis software ( N ≥ 8 biological replicates ) ; data displayed are mean ± SEM . Data was analyzed using 1 - way ANOVA with multiple comparisons ( WM983 , p < 0 . 001 ; UACC257 p > 0 . 05 ) . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . O1C78 WM983 cells expressing the PFO - mCherry D4H cholesterol sensor were examined as in panels H and I ( N ≥ 8 biological replicates ) ; data displayed are mean ± SEM .

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

Figure 4WM983 and UACC257 human melanoma cells were transfected with Perfringolysin O (PFO)-mCherry D4H cholesterol sensor and plated on glass coverslips. Cells were exposed to UV (0 or 175 J/m2) and incubated for 0, 2 or 5 weeks as indicated. (H) Representative cell images. (I) Fluorescence intensity from cells analyzed in (H) was measured and quantified using LASX analysis software (N≥8 biological replicates); data displayed are mean±SEM. Data was analyzed using 1-way ANOVA with multiple comparisons (WM983, p < 0.001; UACC257 p > 0.05). **p<0.01, ***p<0.001, ****p<0.0001.O1C78 WM983 cells expressing the PFO-mCherry D4H cholesterol sensor were examined as in panels H and I (N≥8 biological replicates); data displayed are mean±SEM.


Figure 7Patient surgical explants were received from the biosample repository of FCCC . Cells were isolated from tumors using 50 μM Medicons and the Medimachine system and grown for 3 days before experimentation to eliminate non - melanoma cells . ( A - B ) Plated control or UV irradiated ( 175J / m2 ) cells were then incubated overnight on glass coverslips prior to Fura2 loading and administration of Tg in Ca2 + - free media for 15 minutes . ( A ) Representative examples of SOCE responses in patient samples following the addition of Ca2 + ( 1 mM ) . Each trace represents 5 to 10 individual cells ; solid line represents the mean and the dotted line represents SEM . ( B ) Each dot represents SOCE in cells obtained from a different patient sample ( n = 20 ; data displayed as mean ± SEM ) . The effect of UV on SOCE was determined by one way ANOVA ( p > 0 . 05 ) . ( C ) Comparison of SOCE in cells collected from both primary tumors and metastatic lymph nodes from the same patient ( from patients analyzed in panels A , B ) . ( D ) SOCE in primary patient samples exhibiting NRAS ( 8 of 27 samples ) , BRAF ( 6 of 27 samples ) , TERT promoter ( 6 of 27 samples ) , NF1 ( 5 of 27 samples ) , and CDKN2A ( 5 of 27 samples ) alterations were compared to SOCE in samples that expressed WT or the unaltered gene . Data displayed as mean ± SEM . ( E ) Single cells from patient samples were treated with or without the indicated drug in the presence of Tg in nominally Ca2 + - free media for 15 minutes prior to the addition of Ca2 + ( 1 mM ) . The number on each bar represents the number of patient specimens tested . Data were compared against their matched control analyzed by the unpaired T test ( Tak475 , p = 0 . 015 ; compactin , p = 0 . 0049 ; IA65 , p = 0 . 0390 ) . ( F ) Patient cells were placed in Matrigel transwell inserts in OptiMEM reduced serum media with or without TAK475 ( 10uM ) , compactin ( 2μM ) , IA65 ( 2μM ) or OSMI1 ( 40μM ) ; DMEM with 10 % FBS was used as a chemoattractant . Crystal violet - stained invaded cells were measured 20 hrs after initiation of assay . Each dot represents 1 patient sample . Data analyzed by 2 - way ANOVA ( cholesterol inhibition was p < 0 . 0001 ; patient samples were p < 0 . 001 ; interaction was p < 0 . 01 ) . For IA65 , ( inhibition was p < 0 . 0001 ; patient samples were p < 0 . 01 ; interaction was p < 0 . 0001 ) . For OSMI1 , ( inhibition was p < 0 . 05 ; patient samples were p > 0 . 05 ; interaction was p < 0 . 05 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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

Figure 7Patient surgical explants were received from the biosample repository of FCCC. Cells were isolated from tumors using 50 μM Medicons and the Medimachine system and grown for 3 days before experimentation to eliminate non-melanoma cells. (A-B) Plated control or UV irradiated (175J/m2) cells were then incubated overnight on glass coverslips prior to Fura2 loading and administration of Tg in Ca2+-free media for 15 minutes. (A) Representative examples of SOCE responses in patient samples following the addition of Ca2+ (1 mM). Each trace represents 5 to 10 individual cells; solid line represents the mean and the dotted line represents SEM. (B) Each dot represents SOCE in cells obtained from a different patient sample (n=20; data displayed as mean±SEM). The effect of UV on SOCE was determined by one way ANOVA (p > 0.05). (C) Comparison of SOCE in cells collected from both primary tumors and metastatic lymph nodes from the same patient (from patients analyzed in panels A, B).(D) SOCE in primary patient samples exhibiting NRAS (8 of 27 samples), BRAF (6 of 27 samples), TERT promoter (6 of 27 samples), NF1 (5 of 27 samples), and CDKN2A (5 of 27 samples) alterations were compared to SOCE in samples that expressed WT or the unaltered gene. Data displayed as mean±SEM.(E) Single cells from patient samples were treated with or without the indicated drug in the presence of Tg in nominally Ca2+-free media for 15 minutes prior to the addition of Ca2+ (1 mM). The number on each bar represents the number of patient specimens tested. Data were compared against their matched control analyzed by the unpaired T test (Tak475, p = 0.015; compactin, p = 0.0049; IA65, p = 0.0390).(F) Patient cells were placed in Matrigel transwell inserts in OptiMEM reduced serum media with or without TAK475 (10uM), compactin (2μM), IA65 (2μM) or OSMI1 (40μM); DMEM with 10% FBS was used as a chemoattractant. Crystal violet-stained invaded cells were measured 20 hrs after initiation of assay. Each dot represents 1 patient sample. Data analyzed by 2-way ANOVA (cholesterol inhibition was p < 0.0001; patient samples were p < 0.001; interaction was p < 0.01). For IA65, (inhibition was p < 0.0001; patient samples were p < 0.01; interaction was p < 0.0001). For OSMI1, (inhibition was p < 0.05; patient samples were p > 0.05; interaction was p < 0.05). *p<0.05, **p<0.01, ****p<0.0001.


figf1a , Impairment of two ATG - conjugation systems ( Atg12 and LC3 ) in the Atg7 - deficient brain . Brain homogenates from P28 mice were immunoblotted with antibodies against Atg7 , Atg5 and LC3 . Actin was used as a loading control . Data shown are representative of three separate experiments . b , Kaplan - Meier survival curves of Atg7flox / + ; nestin - Cre ( n = 41 ) and Atg7flox / flox ; nestin - Cre ( n = 26 ) mice over 28 weeks . c , Abnormal limb - clasping reflexes in Atg7flox / flox ; nestin - Cre mice at P28 . When lifted by the tail , Atg7flox / + ; nestin - Cre mice behave normally , extending their hind limbs and bodies . In contrast , Atg7flox / flox ; nestin - Cre mice bend their legs towards their trunk or tighten their back limbs to their bodies and anterior limbs . d , Movement ataxia in Atg7flox / flox ; nestin - Cre mice at P28 . Motor coordination was tested using a rotarod assay . Atg7flox / + ; nestin - Cre ( n = 5 ) and Atg7flox / flox ; nestin - Cre ( n = 5 ) mice were placed on a rod rotating at 20 r . p . m . , and the time spent on the rod was counted . Data show mean ± s . d . * , P 0 . 01 ( Students t - test ) . There was no significant sex difference in survival rate and onset - stage of abnormal mice - clasping and tremor in miceflox / flox ; nestin - Cre mice .

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

figf1a, Impairment of two ATG-conjugation systems (Atg12 and LC3) in the Atg7-deficient brain. Brain homogenates from P28 mice were immunoblotted with antibodies against Atg7, Atg5 and LC3. Actin was used as a loading control. Data shown are representative of three separate experiments.b, Kaplan-Meier survival curves of Atg7flox/+; nestin-Cre (n = 41) and Atg7flox/flox; nestin-Cre (n = 26) mice over 28 weeks.c, Abnormal limb-clasping reflexes in Atg7flox/flox; nestin-Cre mice at P28. When lifted by the tail, Atg7flox/+; nestin-Cre mice behave normally, extending their hind limbs and bodies. In contrast, Atg7flox/flox; nestin-Cre mice bend their legs towards their trunk or tighten their back limbs to their bodies and anterior limbs.d, Movement ataxia in Atg7flox/flox; nestin-Cre mice at P28. Motor coordination was tested using a rotarod assay. Atg7flox/+; nestin-Cre (n = 5) and Atg7flox/flox; nestin-Cre (n = 5) mice were placed on a rod rotating at 20 r.p.m., and the time spent on the rod was counted. Data show mean ± s.d. *, P 0.01 (Students t-test). There was no significant sex difference in survival rate and onset-stage of abnormal mice-clasping and tremor in miceflox/flox; nestin-Cre mice.


figf2a - f , Histological analyses of Atg7flox / + ; nestin - Cre ( left ) and Atg7flox / flox ; nestin - Cre ( right ) cerebral cortex at P56 . Cryosections were stained with HE ( a - d ) or immunostained for the glial marker GFAP ( e , f ) . Boxed areas in a and b are magnified in c and d , respectively . Arrows in c point to large pyramidal neurons in the cerebral cortex . g - j , Histological analysis of Atg7flox / + ; nestin - Cre ( left ) and Atg7flox / flox ; nestin - Cre ( right ) cerebellum at P56 . Cryosections were stained with HE ( g , h ) or immunostained for the Purkinje marker calbindin ( i , j ) . Arrows in g and h indicate cerebellar Purkinje cells . k - p , TUNEL staining of the cerebral cortex ( k , l ) and cerebellum ( n , o ) at P56 in Atg7flox / + ; nestin - Cre ( k , n ) and Atg7flox / flox ; nestin - Cre ( l , o ) sections . TUNEL - positive cells are indicated with arrows and shown as higher magnification images beneath panels l , n and o . Histograms show the average number ( ± s . d . ) of TUNEL - positive cells in ten sections for three animals of each genotype ( m , p ) . * , P 0 . 05 ( t - test ) . Scale bars , 1 mm ( a , b ) , 100 µm ( c - j ) , 250 µm ( k , l , n , o ) . We observed no sex difference in brain morphology or neuronal loss in Atg7flox / flox ; nestin - Cre mice .

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

figf2a-f, Histological analyses of Atg7flox/+; nestin-Cre (left) and Atg7flox/flox; nestin-Cre (right) cerebral cortex at P56. Cryosections were stained with HE (a-d) or immunostained for the glial marker GFAP (e, f). Boxed areas in a and b are magnified in c and d, respectively. Arrows in c point to large pyramidal neurons in the cerebral cortex.g-j, Histological analysis of Atg7flox/+; nestin-Cre (left) and Atg7flox/flox; nestin-Cre (right) cerebellum at P56. Cryosections were stained with HE (g, h) or immunostained for the Purkinje marker calbindin (i, j). Arrows in g and h indicate cerebellar Purkinje cells.k-p, TUNEL staining of the cerebral cortex (k, l) and cerebellum (n, o) at P56 in Atg7flox/+; nestin-Cre (k, n) and Atg7flox/flox; nestin-Cre (l, o) sections. TUNEL-positive cells are indicated with arrows and shown as higher magnification images beneath panels l, n and o. Histograms show the average number (± s.d.) of TUNEL-positive cells in ten sections for three animals of each genotype (m, p). *, P 0.05 (t-test). Scale bars, 1 mm (a, b), 100 µm (c-j), 250 µm (k, l, n, o). We observed no sex difference in brain morphology or neuronal loss in Atg7flox/flox; nestin-Cre mice.


figf3a - l , The presence of ubiquitin - positive dots was examined by immunohistochemistry in several regions including cerebral cortex ( a , b ) , cerebellum ( c , d ) , hippocampus ( e , f ) , hypothalamus ( g , h ) , amygdala ( i , j ) and pontine nuclei ( k , l ) of Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre mice . Note the presence of numerous ubiquitin dots in the amygdala and hypothalamus of the representative mutants . Scale bars , 50 µm . m , n , Electron micrographs of the brain of Atg7flox / flox ; nestin - Cre mice . Inclusion bodies ( arrows ) were often observed in Atg7flox / flox ; nestin - Cre hypothalamus . The boxed region in m is shown in n . Inclusion bodies were not detected in Atg7flox / + ; nestin - Cre brain ( data not shown ) . Scale bars , 5 µm ( m ) , 1 µm ( n ) . o , Immunoelectron micrograph of ubiquitin in a representative Atg7flox / flox ; nestin - Cre hypothalamus . N , nucleus . Scale bar , 1 µm . p , q , Immunohistochemical detection of ubiquitin - positive inclusions in the cerebral cortex ( p ) and hypothalamus ( q ) at P9 , P18 and P56 . Brain sections of each genotype at the indicated ages were immunostained with an anti - ubiquitin antibody . Ubiquitin - positive inclusions appeared at P18 and became larger with ageing in the brain of Atg7flox / flox ; nestin - Cre mice . Scale bars , 50 µm .

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

figf3a-l, The presence of ubiquitin-positive dots was examined by immunohistochemistry in several regions including cerebral cortex (a, b), cerebellum (c, d), hippocampus (e, f), hypothalamus (g, h), amygdala (i, j) and pontine nuclei (k, l) of Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice. Note the presence of numerous ubiquitin dots in the amygdala and hypothalamus of the representative mutants. Scale bars, 50 µm.m, n, Electron micrographs of the brain of Atg7flox/flox; nestin-Cre mice. Inclusion bodies (arrows) were often observed in Atg7flox/flox; nestin-Cre hypothalamus. The boxed region in m is shown in n. Inclusion bodies were not detected in Atg7flox/+; nestin-Cre brain (data not shown). Scale bars, 5 µm (m), 1 µm (n). o, Immunoelectron micrograph of ubiquitin in a representative Atg7flox/flox; nestin-Cre hypothalamus. N, nucleus. Scale bar, 1 µm.p, q, Immunohistochemical detection of ubiquitin-positive inclusions in the cerebral cortex (p) and hypothalamus (q) at P9, P18 and P56. Brain sections of each genotype at the indicated ages were immunostained with an anti-ubiquitin antibody. Ubiquitin-positive inclusions appeared at P18 and became larger with ageing in the brain of Atg7flox/flox; nestin-Cre mice. Scale bars, 50 µm.


figf4a , Increase in ubiquitinated proteins in Atg7flox / flox ; nestin - Cre mouse brain over time . Homogenates of P9 , P18 and P56 brains from Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre mice were immunoblotted with an anti - ubiquitin antibody . An anti - actin antibody was used as a loading control . Data shown are representative of three separate experiments . We observed no sex difference in the accumulation of ubiquitin in Atg7flox / flox ; nestin - Cre mice . b , Peptide hydrolysis activity of 20S and 26Sproteasomes . Homogenates from P28Atg7flox / + ; nestin - Cre ( blue ) and Atg7flox / flox ; nestin - Cre ( pink ) brains were fractionated by glyceroldensity gradient centrifugation ( 10 - 40 % glycerol from fraction 1 to fraction 30 ) . Aliquots from each fraction were used for the assay of chymotryptic activity of proteasomes using Suc - LLVY - AMC as a substrate in the absence ( top ) or presence ( bottom ) of 0 . 05 % SDS . The sedimenting positions of 20S and 26Sproteasomes are indicated with arrowheads . Note that whereas 26Sproteasomes exist in active forms in tissues , 20Sproteasomes are latent and are activated artificially by a low concentration of SDS . c , ATP - dependent degradation of [ 35S ] - labelled ODC . Degradation of [ 35S ] - labelled ODC was assayed using crude extracts from P28 Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre brains . The experiment was repeated three times , and values represent mean ± s . d . In the above assays , there were no significant differences between Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre mice . d , Immunoblot analysis of 26S proteasome components . Homogenates from P28 Atg7flox / flox , Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre brains were immunoblotted with antibodies against the indicated proteins . Data shown are representative of three separate experiments . There was no change in proteasome status for the different genotypes .

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

figf4a, Increase in ubiquitinated proteins in Atg7flox/flox; nestin-Cre mouse brain over time. Homogenates of P9, P18 and P56 brains from Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice were immunoblotted with an anti-ubiquitin antibody. An anti-actin antibody was used as a loading control. Data shown are representative of three separate experiments. We observed no sex difference in the accumulation of ubiquitin in Atg7flox/flox; nestin-Cre mice.b, Peptide hydrolysis activity of 20S and 26Sproteasomes. Homogenates from P28Atg7flox/+; nestin-Cre (blue) and Atg7flox/flox; nestin-Cre (pink) brains were fractionated by glyceroldensity gradient centrifugation (10-40% glycerol from fraction 1 to fraction 30). Aliquots from each fraction were used for the assay of chymotryptic activity of proteasomes using Suc-LLVY-AMC as a substrate in the absence (top) or presence (bottom) of 0.05% SDS. The sedimenting positions of 20S and 26Sproteasomes are indicated with arrowheads. Note that whereas 26Sproteasomes exist in active forms in tissues, 20Sproteasomes are latent and are activated artificially by a low concentration of SDS.c, ATP-dependent degradation of [35S]-labelled ODC. Degradation of [35S]-labelled ODC was assayed using crude extracts from P28 Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains. The experiment was repeated three times, and values represent mean ± s.d. In the above assays, there were no significant differences between Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice.d, Immunoblot analysis of 26S proteasome components. Homogenates from P28 Atg7flox/flox, Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains were immunoblotted with antibodies against the indicated proteins. Data shown are representative of three separate experiments. There was no change in proteasome status for the different genotypes.


Figure 1B - E . Left : At least 150 cells were counted per time point and the number of LD per cell was inspected visually and plotted . The median value of each timepoint is indicated by an orange bar and number . Each panel corresponds to a representative time - course experiment in response to each indicated DNA - damaging agent ( out of at least 3 per agent ) . " unt " = untreated condition . " mock " refers to the culture being treated with DMSO , the dissolving agent for CPT . Used concentrations were 100 µg / mL zeocin , 100 µM CPT , 100 mM HU and 15 µg / mL nocodazole . Right : the same cultures were analysed by cytometry to assess DNA content ( 1C = unreplicated DNA , 2C = fully replicated DNA ) during the time - course to confirm that the used agents were causing cell arrest at the expected phases of the cell cycle . H - K . Cells as described in ( G ) in which the non - induced condition varied between glycerol ( H ) or raffinose ( I - K ) , and the cells harboured either a plasmid driving 59 cuts in the genome ( H - J ) or none ( K ) .

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

Figure 1B-E. Left: At least 150 cells were counted per time point and the number of LD per cell was inspected visually and plotted. The median value of each timepoint is indicated by an orange bar and number. Each panel corresponds to a representative time-course experiment in response to each indicated DNA-damaging agent (out of at least 3 per agent). "unt" = untreated condition. "mock" refers to the culture being treated with DMSO, the dissolving agent for CPT. Used concentrations were 100 µg/mL zeocin, 100 µM CPT, 100 mM HU and 15 µg/mL nocodazole. Right: the same cultures were analysed by cytometry to assess DNA content (1C = unreplicated DNA, 2C = fully replicated DNA) during the time-course to confirm that the used agents were causing cell arrest at the expected phases of the cell cycle.H-K. Cells as described in (G) in which the non-induced condition varied between glycerol (H) or raffinose (I-K), and the cells harboured either a plasmid driving 59 cuts in the genome (H-J) or none (K).


Figure 2B . Exponentially growing S . cerevisiae WT , ste ∆ and tag ∆ cells were treated with 100 µg / mL zeocin and samples collected at the indicated timepoints for western blot analysis and cytometry The activation of the DDR was monitored following the progressive phosphorylation of its downstream effector kinase Rad53 . The unphosphorylated and phosphorylated isoforms of Rad53 are indicated ( Rad53 and Rad53 - P , respectively ) . Ponceau staining is shown as a loading control . The percentage of Rad53 - P was quantified at each point by dividing the raw signal of the upper band by the total signal in that lane , thus plotted in the graph shown on the right . The plotted values represent the mean value of at least 3 independent experiments and the variation is represented by the SEM . Unpaired t - tests were used to compare the potential differences of the means at each time point . Only the p - value ( s ) for those being significantly different are indicated . C . Identical to ( B ) but comparing unloaded WT and WT pre - loaded for 2 h with 0 . 05 % oleate in order to inhibit sterol esterification . D . Non - confluent RPE - 1 and Huh - 7 human cell lines were either left untreated , or treated for 2 h with the DNA - damaging agents MMS ( 0 . 005 % ) or zeocin ( 10 µg / mL ) . Prior to that , cells were pre - loaded with oleate ( 4 h at 60 µM ) to inhibit sterol esterification or treated with 30 µM BSA ( mock ) as a control . The downstream effector kinase of the DDR CHK2 was monitored by western blot for its activation using a specific antibody against its phosphorylation at Thr68 ( P - CHK2 ) . The western blot signal for total CHK2 , calnexin and the Ponceau staining are used as loading controls . The bars in the graph show the mean value of the P - CHK2 to CHK2 signals ratio for genotoxins - treated cells while the dots correspond to the individual values out of 3 independent experiments . E . Identical to ( D ) though only in response to zeocin and by inhibiting sterols with the specific inhibitor avasimibe ( 5 µM for 2 hours ) . Plot details as in ( D ) .

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

Figure 2B. Exponentially growing S. cerevisiae WT, ste∆ and tag∆ cells were treated with 100 µg/mL zeocin and samples collected at the indicated timepoints for western blot analysis and cytometry The activation of the DDR was monitored following the progressive phosphorylation of its downstream effector kinase Rad53. The unphosphorylated and phosphorylated isoforms of Rad53 are indicated (Rad53 and Rad53-P, respectively). Ponceau staining is shown as a loading control. The percentage of Rad53-P was quantified at each point by dividing the raw signal of the upper band by the total signal in that lane, thus plotted in the graph shown on the right. The plotted values represent the mean value of at least 3 independent experiments and the variation is represented by the SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated. C. Identical to (B) but comparing unloaded WT and WT pre-loaded for 2 h with 0.05% oleate in order to inhibit sterol esterification.D. Non-confluent RPE-1 and Huh-7 human cell lines were either left untreated, or treated for 2 h with the DNA-damaging agents MMS (0.005%) or zeocin (10 µg/mL). Prior to that, cells were pre-loaded with oleate (4 h at 60 µM) to inhibit sterol esterification or treated with 30 µM BSA (mock) as a control. The downstream effector kinase of the DDR CHK2 was monitored by western blot for its activation using a specific antibody against its phosphorylation at Thr68 (P-CHK2). The western blot signal for total CHK2, calnexin and the Ponceau staining are used as loading controls. The bars in the graph show the mean value of the P-CHK2 to CHK2 signals ratio for genotoxins-treated cells while the dots correspond to the individual values out of 3 independent experiments. E. Identical to (D) though only in response to zeocin and by inhibiting sterols with the specific inhibitor avasimibe (5 µM for 2 hours). Plot details as in (D).


Figure 3A . Exponentially growing S . cerevisiae WT and yeh2 ∆ cells were treated with 100 µg / mL zeocin and samples retrieved at the indicated timepoints for Western blot analysis and cytometry The activation of the DDR was monitored following the progressive phosphorylation of its downstream effector kinase Rad53 . The unphosphorylated and phosphorylated isoforms of Rad53 are indicated ( Rad53 and Rad53 - P , respectively ) . Ponceau staining is shown as a loading control . The percentage of Rad53 - P was quantified at each timepoint and plotted in the graph shown on the right . The plotted values represent the mean value of at least 3 independent experiments and the variation is represented as the SEM . Unpaired t - tests were used to compare the potential differences of the means at each time point . Only the p - value ( s ) for those being significantly different are indicated . C . Exponentially growing S . cerevisiae WT and ste ∆ cells were treated with 100 µg / mL zeocin and samples retrieved at the indicated timepoints for inspection by fluorescence microscopy . The establishment of DNA resection factories was assessed by counting the number of Rfa1 - CFP foci per cell . Scale bar is 4 µm . Left : the mean values of each timepoint of three independent experiments were used to obtain this graph ( mean and SEM ) . Unpaired t - tests were used to compare the potential differences of the means at each time point . Only the p - value ( s ) for those being significantly different are indicated . Right : representative images of both strains at timepoint 120 min . Scale bar is 4 µm . D . WT and yeh2 ∆ cells carrying a plasmid bearing the HO nuclease gene under a galactose - inducible promoter were exposed to 2 % galactose to trigger the expression of the HO nuclease to cut at the MAT locus , and samples were retrieved at the indicated time points for genomic DNA extraction . Subsequent SspI digestion , in combination with the use of a probe targeted to MAT , allows defining the fate of the cut fragment by Southern blot analysis . As resection progresses on DNA , SspI restriction sites are lost , leading to progressively longer ssDNA fragments ( whose sizes are indicated at the right of the gel ) that can be separated on an agarose gel under denaturing conditions . Given the much stronger signal of the " cut " fragments with respect to the " resected " fragments , the gel has been split into low and high contrast halves , respectively . The ssDNA over cut yet unresected DSB molecules was calculated for each shown resection intermediate . The graph on the right plots the sum of all these values ( cumulative ssDNA / DSB ) for a given resected intermediate during the interval from 90 to 240 min . The error bar is the SEM of three independent experiments . * derives from applying a t - test that compares the two populations of values . E . Cells were pre - loaded with 60 µM oleate 30 µM BSA ( mock ) for 4 h , and then exposed to 0 . 005 % MMS for the indicated times . Total genomic DNA was extracted ( left panel ) and 1500 ng of each condition were spotted onto a nylon membrane directly . The DNA , after being crosslinked to the membrane , was subjected to detection using an anti - ssDNA antibody ( middle panel ) . The raw ssDNA signals obtained this way were quantified for three independent kinetics . The graph ( right panel ) shows the mean raw ssDNA signal value and the error bars are the SEM . Unpaired t - tests were used to compare the potential differences of the means at each time point . Only the p - value ( s ) for those being significantly different are indicated . As a control for the specificity of the anti - ssDNA antibody , we used the sample " MMS 3 h " for treatment with Mung Bean Nuclease ( MBN ) , in order to digest single stranded DNA ( ssDNA ) , or for denaturation , by addition of NaOH to a final 0 . 4 N concentration . These three samples are shown in the top part of the membrane .

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

Figure 3A. Exponentially growing S. cerevisiae WT and yeh2∆ cells were treated with 100 µg/mL zeocin and samples retrieved at the indicated timepoints for Western blot analysis and cytometry The activation of the DDR was monitored following the progressive phosphorylation of its downstream effector kinase Rad53. The unphosphorylated and phosphorylated isoforms of Rad53 are indicated (Rad53 and Rad53-P, respectively). Ponceau staining is shown as a loading control. The percentage of Rad53-P was quantified at each timepoint and plotted in the graph shown on the right. The plotted values represent the mean value of at least 3 independent experiments and the variation is represented as the SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated.C. Exponentially growing S. cerevisiae WT and ste∆ cells were treated with 100 µg/mL zeocin and samples retrieved at the indicated timepoints for inspection by fluorescence microscopy. The establishment of DNA resection factories was assessed by counting the number of Rfa1-CFP foci per cell. Scale bar is 4 µm. Left: the mean values of each timepoint of three independent experiments were used to obtain this graph (mean and SEM). Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated. Right: representative images of both strains at timepoint 120 min. Scale bar is 4 µm.D. WT and yeh2∆ cells carrying a plasmid bearing the HO nuclease gene under a galactose-inducible promoter were exposed to 2% galactose to trigger the expression of the HO nuclease to cut at the MAT locus, and samples were retrieved at the indicated time points for genomic DNA extraction. Subsequent SspI digestion, in combination with the use of a probe targeted to MAT, allows defining the fate of the cut fragment by Southern blot analysis. As resection progresses on DNA, SspI restriction sites are lost, leading to progressively longer ssDNA fragments (whose sizes are indicated at the right of the gel) that can be separated on an agarose gel under denaturing conditions. Given the much stronger signal of the "cut" fragments with respect to the "resected" fragments, the gel has been split into low and high contrast halves, respectively. The ssDNA over cut yet unresected DSB molecules was calculated for each shown resection intermediate. The graph on the right plots the sum of all these values (cumulative ssDNA/DSB) for a given resected intermediate during the interval from 90 to 240 min. The error bar is the SEM of three independent experiments. * derives from applying a t-test that compares the two populations of values.E. Cells were pre-loaded with 60 µM oleate 30 µM BSA (mock) for 4 h, and then exposed to 0.005% MMS for the indicated times. Total genomic DNA was extracted (left panel) and 1500 ng of each condition were spotted onto a nylon membrane directly. The DNA, after being crosslinked to the membrane, was subjected to detection using an anti-ssDNA antibody (middle panel). The raw ssDNA signals obtained this way were quantified for three independent kinetics. The graph (right panel) shows the mean raw ssDNA signal value and the error bars are the SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated. As a control for the specificity of the anti-ssDNA antibody, we used the sample "MMS 3 h" for treatment with Mung Bean Nuclease (MBN), in order to digest single stranded DNA (ssDNA), or for denaturation, by addition of NaOH to a final 0.4 N concentration. These three samples are shown in the top part of the membrane.


Figure 4B . Recombination assay details as in ( A ) using WT , rad3 - 102 , ste ∆ and rad3 - 102 ste ∆ strains . C , D . Pulsed Field Gel Electrophoresis ( PFGE ) performed on DNA samples prepared from RPE - 1 or Huh - 7 cells that had been treated ( or not ) with zeocin and / or oleate ( 2 hours at 10 µg / mL zeocin ; pre - treatments for 4 hours with 60 µM oleate ) to evaluate the presence of DSBs under such treatments . Intact DNA molecules remain in the well , while broken molecules migrate into the gel . Given the action of zeocin , in - gel molecules can be subdivided in high - molecular weight fragments ( indicated by " HMW " ) and low - molecular weight ones ( LMW ) . Plugs were prepared at 37 ° C to prevent artefactual breaking of DNA molecules The bottom graphs provide the percentage of total broken molecules per lane with respect to all molecules in that lane . 3 - 4 independent experiments are plotted , and their median value indicated by an orange bar . The lateral graphs provide the relative intensity ( arbitrary units ) of signals associated with in - gel broken molecules . The values corresponding to zeocin - treated samples are displayed in pink , those corresponding to oleate + zeocin - treated samples in orange . For pink and orange curves , the mean value of 3 - 4 independent experiments is displayed in dark colour , while the SEM is indicated in light colours . Statistical analyses of the difference between treatments was assessed using a t - test .

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

Figure 4B. Recombination assay details as in (A) using WT, rad3-102, ste∆ and rad3-102 ste∆ strains.C, D. Pulsed Field Gel Electrophoresis (PFGE) performed on DNA samples prepared from RPE-1 or Huh-7 cells that had been treated (or not) with zeocin and/or oleate (2 hours at 10 µg/mL zeocin; pre-treatments for 4 hours with 60 µM oleate) to evaluate the presence of DSBs under such treatments. Intact DNA molecules remain in the well, while broken molecules migrate into the gel. Given the action of zeocin, in-gel molecules can be subdivided in high-molecular weight fragments (indicated by "HMW") and low-molecular weight ones (LMW). Plugs were prepared at 37°C to prevent artefactual breaking of DNA molecules The bottom graphs provide the percentage of total broken molecules per lane with respect to all molecules in that lane. 3-4 independent experiments are plotted, and their median value indicated by an orange bar. The lateral graphs provide the relative intensity (arbitrary units) of signals associated with in-gel broken molecules. The values corresponding to zeocin-treated samples are displayed in pink, those corresponding to oleate + zeocin-treated samples in orange. For pink and orange curves, the mean value of 3-4 independent experiments is displayed in dark colour, while the SEM is indicated in light colours. Statistical analyses of the difference between treatments was assessed using a t-test.


Figure 5A . Exponentially growing S . cerevisiae WT , ste ∆ , tag ∆ and yeh2 ∆ cells were treated with 100 µg / mL zeocin for 90 minutes . This treatment duration recurrently results in more cells accumulating in the G1 phase of the cell cycle After 90 min , cells were washed to remove zeocin and resuspended in fresh , zeocin - free medium , and samples were retrieved every hour , as indicated . All samples were processed for western blot analysis and cytometry . The de - activation of the DDR was monitored following the progressive de - phosphorylation of Rad53 . The unphosphorylated and phosphorylated isoforms of Rad53 are indicated ( Rad53 and Rad53 - P , respectively ) . Ponceau staining is shown as a loading control . Top : samples were migrated using commercial 3 - 8 % gradient gels . Bottom : samples were migrated in home - made resolutive gels ( see M & M ) . B . The percentage of Rad53 - P was quantified at each time point as in Fig 2B . The plotted values represent the mean value of at least 3 independent experiments and the variability is represented by the SEM . Unpaired one - way ANOVA tests were conducted to compare the potential differences of the means of the four conditions at each time point . Only the p - value ( s ) for the comparisons being significantly different are indicated , where * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 .

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

Figure 5A. Exponentially growing S. cerevisiae WT, ste∆, tag∆ and yeh2∆ cells were treated with 100 µg/mL zeocin for 90 minutes. This treatment duration recurrently results in more cells accumulating in the G1 phase of the cell cycle After 90 min, cells were washed to remove zeocin and resuspended in fresh, zeocin-free medium, and samples were retrieved every hour, as indicated. All samples were processed for western blot analysis and cytometry. The de-activation of the DDR was monitored following the progressive de-phosphorylation of Rad53. The unphosphorylated and phosphorylated isoforms of Rad53 are indicated (Rad53 and Rad53-P, respectively). Ponceau staining is shown as a loading control. Top: samples were migrated using commercial 3-8% gradient gels. Bottom: samples were migrated in home-made resolutive gels (see M&M).B. The percentage of Rad53-P was quantified at each time point as in Fig 2B. The plotted values represent the mean value of at least 3 independent experiments and the variability is represented by the SEM. Unpaired one-way ANOVA tests were conducted to compare the potential differences of the means of the four conditions at each time point. Only the p-value(s) for the comparisons being significantly different are indicated, where *, p < 0.05; **, p < 0.01; ****, p < 0.0001.


Figure 6B . Details as in ( A ) but for the indicated strains . A one - way ANOVA for multiple comparisons against the WT values ( not plotted ) was applied to evaluate whether there were significant differences between the means of the 210 time - point . p - values are indicated by asterisks . * , p < 0 . 05 . C . Left : Representative images obtained after immunofluorescence to detect P - ATM ( ATM phosphorylation at Serine 1981 ) signal in RPE - 1 and Huh - 7 fixed cells that had been treated ( or not ) with 0 . 005 % MMS or 10 µg / mL zeocin for 2 hours ; pre - treatments were done for 4 hours with 60 µM oleate . Size bars are 10 µm . Right : Quantification of the percentage of P - ATM - positive cells . The values obtained from 3 to 4 independent experiments are plotted as black spots , and the mean of them is shown as an orange bar . At least 200 cells were counted per condition and experiment . The p - values obtained after performing an unpaired t - test are shown . D . RPE - 1 and Huh - 7 cells were treated or not with MMS 0 . 005 % for 2 hours . Thereafter , the ATM inhibitor AZD0156 was added at a final concentration of 1 , 10 or 50 nM for 1 hour . Cells were collected and phosphorylation on Thr68 of CHK2 and tubulin levels were analyzed by western blot . E . Details as in ( C ) but to detect phosphorylated H2AX ( yH2AX ) . The graph shows the mean fold - change in signal per nucleus out of at least 300 nuclei counted per condition and experiment . 3 to 4 independent experiments were performed . The bar height indicates the value of the mean of the means and , the error bars , the associated SEM .

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

Figure 6B. Details as in (A) but for the indicated strains. A one-way ANOVA for multiple comparisons against the WT values (not plotted) was applied to evaluate whether there were significant differences between the means of the 210 time-point. p-values are indicated by asterisks. *, p < 0.05.C. Left: Representative images obtained after immunofluorescence to detect P-ATM (ATM phosphorylation at Serine 1981) signal in RPE-1 and Huh-7 fixed cells that had been treated (or not) with 0.005% MMS or 10 µg/mL zeocin for 2 hours; pre-treatments were done for 4 hours with 60 µM oleate. Size bars are 10 µm. Right: Quantification of the percentage of P-ATM-positive cells. The values obtained from 3 to 4 independent experiments are plotted as black spots, and the mean of them is shown as an orange bar. At least 200 cells were counted per condition and experiment. The p-values obtained after performing an unpaired t-test are shown.D. RPE-1 and Huh-7 cells were treated or not with MMS 0.005% for 2 hours. Thereafter, the ATM inhibitor AZD0156 was added at a final concentration of 1, 10 or 50 nM for 1 hour. Cells were collected and phosphorylation on Thr68 of CHK2 and tubulin levels were analyzed by western blot.E. Details as in (C) but to detect phosphorylated H2AX (yH2AX). The graph shows the mean fold-change in signal per nucleus out of at least 300 nuclei counted per condition and experiment. 3 to 4 independent experiments were performed. The bar height indicates the value of the mean of the means and, the error bars, the associated SEM.


Figure 7B . A hydrophobic membrane on which 100 pmol of the indicated lipid species were spotted was incubated with immunopurified FLAG - Tel1 and further developed using an anti - FLAG antibody . The bar lengths in the graph represent the mean value of 4 independent experiments and the error bars , the associated SEM . To assess whether the mean values differed significantly , a t - test was performed and p - values are shown . Additionally , an identical lipid - containing membrane was incubated with 3xFLAG peptide and subsequently developed using an anti - FLAG antibody to control that the observed signals were not due to FLAG peptide binding alone . PI ; phosphatidylinositol ; PI ( 4 ) P , phosphatidylinositol - 4 - phosphate ; PI ( 4 , 5 ) P , phosphatidylinositol - 4 , 5 - bisphosphate ; PI ( 3 , 4 , 5 ) P , phosphatidylinositol - 3 , 4 , 5 - triphosphate . D . Proximity ligation assay ( PLA ) to assess whether ATM and PI ( 4 ) P are in close proximity ( < 40 nm ) . Top : representative pictures of RPE - 1 cells , in which PLA signals are pictured in green . Size bar is 10 µm . Bottom left : the graph shows the mean PLA signal area occupied per cell when doing the experiment with both antibodies ( experiment ) or by omitting one primary antibody at a time ( technical controls ) . In practice , each point is the value of having measured all the PLA signals present in one photo ( 20 to 40 cells ) and divided this value by the number of nuclei . To account for reproducibility , we used SuperPlots to draw the graphs each independent experiment is plotted in a different colour , where the mean value of each independent experiment is highlighted by a more intense colour than the individual values of that experiment , for which the colour is more translucid . Last , the solid horizontal line marks the mean of the means . A one - way ANOVA for multiple comparisons was applied to evaluate whether there were significant differences between the means . Bottom right : the graph shows the mean PLA signal area occupied per cell when doing the experiment either in cells transfected with an siRNA Control ( experiment ) or with three different siRNAs targeted against ATM ( biological controls ) . The meaning of each plotted point , of the horizontal line and the statistical analysis is as explained above .

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

Figure 7B. A hydrophobic membrane on which 100 pmol of the indicated lipid species were spotted was incubated with immunopurified FLAG-Tel1 and further developed using an anti-FLAG antibody. The bar lengths in the graph represent the mean value of 4 independent experiments and the error bars, the associated SEM. To assess whether the mean values differed significantly, a t-test was performed and p-values are shown. Additionally, an identical lipid-containing membrane was incubated with 3xFLAG peptide and subsequently developed using an anti-FLAG antibody to control that the observed signals were not due to FLAG peptide binding alone. PI; phosphatidylinositol; PI(4)P, phosphatidylinositol-4-phosphate; PI(4,5)P, phosphatidylinositol-4,5-bisphosphate; PI(3,4,5)P, phosphatidylinositol-3,4,5-triphosphate.D. Proximity ligation assay (PLA) to assess whether ATM and PI(4)P are in close proximity (< 40 nm). Top: representative pictures of RPE-1 cells, in which PLA signals are pictured in green. Size bar is 10 µm. Bottom left: the graph shows the mean PLA signal area occupied per cell when doing the experiment with both antibodies (experiment) or by omitting one primary antibody at a time (technical controls). In practice, each point is the value of having measured all the PLA signals present in one photo (20 to 40 cells) and divided this value by the number of nuclei. To account for reproducibility, we used SuperPlots to draw the graphs each independent experiment is plotted in a different colour, where the mean value of each independent experiment is highlighted by a more intense colour than the individual values of that experiment, for which the colour is more translucid. Last, the solid horizontal line marks the mean of the means. A one-way ANOVA for multiple comparisons was applied to evaluate whether there were significant differences between the means. Bottom right: the graph shows the mean PLA signal area occupied per cell when doing the experiment either in cells transfected with an siRNA Control (experiment) or with three different siRNAs targeted against ATM (biological controls). The meaning of each plotted point, of the horizontal line and the statistical analysis is as explained above.


Figure 8B . Left : RPE - 1 cells untreated or exposed to 10 µg / mL zeocin for 2 h were used either for whole - cell protein extraction ( W ) or for fractionation into cytoplasmic ( C ) and nuclear ( N ) fractions in order to assess relative ATM distribution . Fractionation quality was assessed by checking for histone 3 ( H3 ) ( nucleus ) and GOLPH3 ( cytoplasm ) signals . Right : To quantify the relative distribution of total ATM between nucleus and cytoplasm , total ATM signals were normalized to total Ponceau signals from the corresponding fraction . Each dot of a given color belongs to an independent experiment , the bar height shows the mean of those experiments , and the error bars represent the SEM . The p - value is derived from an unpaired t - test . D . Exponentially growing S . cerevisiae WT cells transformed with an empty vector ( " control " ) or with a plasmid overexpressing OSH4 were treated with 100 µg / mL zeocin and samples collected at the indicated timepoints for inspection by fluorescence microscopy to evaluate the percentage of cells displaying GFP - Tel1 foci . Top : illustrative images of GFP channel ( nuclear Tel1 signals ) in the indicated conditions . Note that we systematically monitor mCherry - Pus1 to ascertain whether signals are nuclear or not . Scale bar is 6 µm . Bottom : graph displaying the mean value of the percentage of cells forming GFP - Tel1 foci and the SEM of 3 independent experiments for the indicated conditions . Unpaired t - tests were used to compare the potential differences of the means at each time point . Only the p - value ( s ) for those being significantly different are indicated .

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

Figure 8B. Left: RPE-1 cells untreated or exposed to 10 µg/mL zeocin for 2 h were used either for whole-cell protein extraction (W) or for fractionation into cytoplasmic (C) and nuclear (N) fractions in order to assess relative ATM distribution. Fractionation quality was assessed by checking for histone 3 (H3) (nucleus) and GOLPH3 (cytoplasm) signals. Right: To quantify the relative distribution of total ATM between nucleus and cytoplasm, total ATM signals were normalized to total Ponceau signals from the corresponding fraction. Each dot of a given color belongs to an independent experiment, the bar height shows the mean of those experiments, and the error bars represent the SEM. The p-value is derived from an unpaired t-test.D. Exponentially growing S. cerevisiae WT cells transformed with an empty vector ("control") or with a plasmid overexpressing OSH4 were treated with 100 µg/mL zeocin and samples collected at the indicated timepoints for inspection by fluorescence microscopy to evaluate the percentage of cells displaying GFP-Tel1 foci. Top: illustrative images of GFP channel (nuclear Tel1 signals) in the indicated conditions. Note that we systematically monitor mCherry-Pus1 to ascertain whether signals are nuclear or not. Scale bar is 6 µm. Bottom: graph displaying the mean value of the percentage of cells forming GFP-Tel1 foci and the SEM of 3 independent experiments for the indicated conditions. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated.


Figure 9A . Western blot showing P - ATM ( ATM phosphorylation at Serine 1981 ) detection . Calnexin is used as a loading control The experiment assesses the ability of the ATM inhibitor AZD0156 ( ATMi ) or the OSBP1 inhibitor schweinfurthin G ( SWG ) to extinguish P - ATM signals triggered by the previous exposure to different genotoxins . Thus , RPE - 1 cells were treated either with 0 . 005 % MMS , or 10 µg / mL zeocin , or 120 ng / mL neocarzinostatin ( NCZ ) , or 200 µM camptothecin for 2 h , then 500 nM AZD156 ( ATMi ) or 10 nM SWG were added in the sustained presence of the genotoxins for 2 additional hours . The images are illustrative of three to four independent experiments whose quantifications are shown in the bottom . Each point illustrates the observed raw P - ATM signal for each condition in each experiment , and similarly coloured dots belong to a same experiment . All the signals belonging to a same experiment were obtained from simultaneously run and transferred gels and simultaneously hybridized and exposed membranes . Thick dashed black lines represent the median of the experiments , and their specific value is shown as a red number on top of each violin plot , while thin dashed black lines indicate first and third quartiles . B . Top : RPE - 1 cells transfected after 3 days with siRNA control or against OSBP1 ( 3 different siOSBP1 sequences were assessed independently ) were exposed to 0 . 005 % MMS for the indicated times and the phosphorylation status of KAP - 1 on Ser 824 was assessed by western blot . Total KAP - 1 signals and the Ponceau staining are used as loading controls . Bottom : graph displaying the P - KAP - 1 / KAP - 1 ratio . Each plotted value is the mean out of three independent experiments , and the error bars account for the SEM . The displayed p - value highlights the significant difference of the concerned means after applying an unpaired t - test . D . RPE - 1 cells were grown with 10 µM BrdU in the culture medium for the last 16 h prior to further processing . Cells were then either mock - treated , or pre - treated for 1 h with 500 nM of the ATM inhibitor ( ATMi ) AZD0156 or 10 nM of the specific OSBP1 inhibitor schweinfurthin G ( SWG ) . Then , 200 µM CPT was added and cells were processed for BrdU detection at the indicated time points . Left : an illustrative image of BrdU - positive nuclei , indicated by yellow arrowheads . Scale bar is 15 µm . Right : the percentage of cells in the population whose nuclei were positive for BrdU was established by visual inspection of the acquired images . The graph shows the mean percentage out of three independent experiments and the error bars indicate their associated SEM . Unpaired t - tests were used to compare the potential differences of the means at each time point . Only the p - value for the one being significantly different is indicated .

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

Figure 9A. Western blot showing P-ATM (ATM phosphorylation at Serine 1981) detection. Calnexin is used as a loading control The experiment assesses the ability of the ATM inhibitor AZD0156 (ATMi) or the OSBP1 inhibitor schweinfurthin G (SWG) to extinguish P-ATM signals triggered by the previous exposure to different genotoxins. Thus, RPE-1 cells were treated either with 0.005% MMS, or 10 µg/mL zeocin, or 120 ng/mL neocarzinostatin (NCZ), or 200 µM camptothecin for 2 h, then 500 nM AZD156 (ATMi) or 10 nM SWG were added in the sustained presence of the genotoxins for 2 additional hours. The images are illustrative of three to four independent experiments whose quantifications are shown in the bottom. Each point illustrates the observed raw P-ATM signal for each condition in each experiment, and similarly coloured dots belong to a same experiment. All the signals belonging to a same experiment were obtained from simultaneously run and transferred gels and simultaneously hybridized and exposed membranes. Thick dashed black lines represent the median of the experiments, and their specific value is shown as a red number on top of each violin plot, while thin dashed black lines indicate first and third quartiles.B. Top: RPE-1 cells transfected after 3 days with siRNA control or against OSBP1 (3 different siOSBP1 sequences were assessed independently) were exposed to 0.005% MMS for the indicated times and the phosphorylation status of KAP-1 on Ser 824 was assessed by western blot. Total KAP-1 signals and the Ponceau staining are used as loading controls. Bottom: graph displaying the P-KAP-1 / KAP-1 ratio. Each plotted value is the mean out of three independent experiments, and the error bars account for the SEM. The displayed p-value highlights the significant difference of the concerned means after applying an unpaired t-test.D. RPE-1 cells were grown with 10 µM BrdU in the culture medium for the last 16 h prior to further processing. Cells were then either mock-treated, or pre-treated for 1 h with 500 nM of the ATM inhibitor (ATMi) AZD0156 or 10 nM of the specific OSBP1 inhibitor schweinfurthin G (SWG). Then, 200 µM CPT was added and cells were processed for BrdU detection at the indicated time points. Left: an illustrative image of BrdU-positive nuclei, indicated by yellow arrowheads. Scale bar is 15 µm. Right: the percentage of cells in the population whose nuclei were positive for BrdU was established by visual inspection of the acquired images. The graph shows the mean percentage out of three independent experiments and the error bars indicate their associated SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value for the one being significantly different is indicated.


Figure 1Sanger - sequencing chromatograms showing the target region of sgAkt1E17K in wild - type ( WT ) and base edited ( BE ) cells . Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3 - expressing NIH3T3 cells with Lenti - sgAkt1E17K . EditR ( Kluesner et al . , 2018 ) was used to calculate the frequency ( % ) of C - to - T conversion at C7 of the protospacer targeted by sgAkt1E17K in BE3 - expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors . Kaplan - Meier curves showing mammary tumor - specific survival for the different models . WB1P - BE3 females injected with Lenti - sgAkt1E17K - Myc ( n = 12 ) showed a reduced mammary tumor - specific survival compared to WB1P - BE3 female mice injected with Lenti - sgNT - Myc ( n = 11 ) vectors ( 58 days after injection vs 72 days after injection , P < 0 . 01 by Mantel - Cox test ) . Sanger - sequencing chromatograms showing the target region of sgAkt1E17K in 3 independent tumors from WB1P - BE3 females injected with Lenti - sgAkt1E17K - Myc . Arrowheads highlight cytosines of the protospacer that show base editing . EditR was used to calculate the average frequency ( % ) of C - to - T conversion at C7 of the protospacer in tumors from WB1P - BE3 females injected with Lenti - sgNT - Myc or Lenti - sgAkt1E17K - Myc .

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

Figure 1Sanger-sequencing chromatograms showing the target region of sgAkt1E17K in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgAkt1E17K. EditR (Kluesner et al., 2018) was used to calculate the frequency (%) of C-to-T conversion at C7 of the protospacer targeted by sgAkt1E17K in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgAkt1E17K-Myc (n=12) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (58 days after injection vs 72 days after injection, P < 0.01 by Mantel-Cox test).Sanger-sequencing chromatograms showing the target region of sgAkt1E17K in 3 independent tumors from WB1P-BE3 females injected with Lenti-sgAkt1E17K-Myc. Arrowheads highlight cytosines of the protospacer that show base editing. EditR was used to calculate the average frequency (%) of C-to-T conversion at C7 of the protospacer in tumors from WB1P-BE3 females injected with Lenti-sgNT-Myc or Lenti-sgAkt1E17K-Myc.


Figure 2Sanger - sequencing chromatograms showing the target regions of sgPik3caE542K , sgPik3caE545K and sgPik3caE453K in wild - type ( WT ) and base edited ( BE ) cells . Arrowheads highlight cytosines of the protospacers that show base editing 5 days after transduction of BE3 - expressing NIH3T3 cells with Lenti - sgPik3caE542K , Lenti - sgPik3caE545K and Lenti - sgPik3caE453K . EditR was used to calculate the frequency ( % ) of C - to - T conversion at the indicated target cytosines of the protospacers in BE3 - expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors . Kaplan - Meier curves showing mammary tumor - specific survival for the different models . WB1P - BE3 females injected with Lenti - sgPik3caE542K - Myc ( n = 10 ) , Lenti - sgPik3caE545K - Myc ( n = 10 ) and Lenti - sgPik3caE453K - Myc ( n = 10 ) showed a reduced mammary tumor - specific survival compared to WB1P - BE3 female mice injected with Lenti - sgNT - Myc ( n = 11 ) vectors ( respectively 47 , 44 and 49 days after injection vs 72 days after injection , P < 0 . 0001 by Mantel - Cox test ) . Sanger - sequencing chromatograms showing the target region of sgPik3caE542K , sgPik3caE545K and sgPik3caE453K in 3 independent tumors from WB1P - BE3 females injected with the corresponding Lenti - sgPik3ca - Myc vectors . Arrowheads highlight cytosines of the protospacer that show base editing . EditR was used to calculate the average frequency ( % ) of C - to - T conversion at the indicated target cytosines of the protospacers in tumors from WB1P - BE3 females injected with Lenti - sgNT - Myc or Lenti - sgPik3caE542K - Myc , Lenti - sgPik3caE545K - Myc and Lenti - sgPik3caE453K - Myc .

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

Figure 2Sanger-sequencing chromatograms showing the target regions of sgPik3caE542K, sgPik3caE545K and sgPik3caE453K in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacers that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgPik3caE542K, Lenti-sgPik3caE545K and Lenti-sgPik3caE453K.EditR was used to calculate the frequency (%) of C-to-T conversion at the indicated target cytosines of the protospacers in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgPik3caE542K-Myc (n=10), Lenti-sgPik3caE545K-Myc (n=10) and Lenti-sgPik3caE453K-Myc (n=10) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (respectively 47, 44 and 49 days after injection vs 72 days after injection, P < 0.0001 by Mantel-Cox test).Sanger-sequencing chromatograms showing the target region of sgPik3caE542K, sgPik3caE545K and sgPik3caE453K in 3 independent tumors from WB1P-BE3 females injected with the corresponding Lenti-sgPik3ca-Myc vectors. Arrowheads highlight cytosines of the protospacer that show base editing. EditR was used to calculate the average frequency (%) of C-to-T conversion at the indicated target cytosines of the protospacers in tumors from WB1P-BE3 females injected with Lenti-sgNT-Myc or Lenti-sgPik3caE542K-Myc, Lenti-sgPik3caE545K-Myc and Lenti-sgPik3caE453K-Myc.


Figure 3Sanger - sequencing chromatograms showing the target region of sgPtenQ245 * in wild - type ( WT ) and base edited ( BE ) cells . Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3 - expressing NIH3T3 cells with Lenti - sgPtenQ245 * . EditR was used to calculate the frequency ( % ) of C - to - T conversion at C4 of the protospacer targeted by sgPtenQ245 * in BE3 - expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors . Kaplan - Meier curves showing mammary tumor - specific survival for the different models . WB1P - BE3 females injected with Lenti - sgPtenQ245 * - Myc ( n = 11 ) showed a reduced mammary tumor - specific survival compared to WB1P - BE3 female mice injected with Lenti - sgNT - Myc ( n = 11 ) vectors ( 37 days after injection vs 72 days after injection , P < 0 . 0001 by Mantel - Cox test ) . BE Analyzer ( Hwang et al . , 2018 ) was used to assess from next - generation sequencing data the fraction of wild - type Pten alleles , base edited alleles or alleles with insertions / deletions ( indels ) in tumors from WB1P - BE3 animals injected with Lenti - sgNT - Myc or Lenti - sgPtenQ245 * - Myc . TIDE analysis showing the spectrum of indels of the targeted Pten alleles in two independent representative tumors from WB1P - BE3 mice injected with Lenti - sgPtenQ245 * - Myc . For the two tumors shown in ( F ) , Sanger - sequencing chromatograms showing the target region of sgPtenQ245 * ( PCR products were subcloned for clarity ) . Arrowheads highlight cytosines of the protospacer that show base editing . In the lower example the gene was inactivated by indels at both alleles , while in the upper one by Q245 * base editing in one allele and a deletion at the second copy of the gene .

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

Figure 3Sanger-sequencing chromatograms showing the target region of sgPtenQ245* in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgPtenQ245*. EditR was used to calculate the frequency (%) of C-to-T conversion at C4 of the protospacer targeted by sgPtenQ245* in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgPtenQ245*-Myc (n=11) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (37 days after injection vs 72 days after injection, P < 0.0001 by Mantel-Cox test).BE Analyzer (Hwang et al., 2018) was used to assess from next-generation sequencing data the fraction of wild-type Pten alleles, base edited alleles or alleles with insertions/deletions (indels) in tumors from WB1P-BE3 animals injected with Lenti-sgNT-Myc or Lenti-sgPtenQ245*-Myc. TIDE analysis showing the spectrum of indels of the targeted Pten alleles in two independent representative tumors from WB1P-BE3 mice injected with Lenti-sgPtenQ245*-Myc. For the two tumors shown in (F), Sanger-sequencing chromatograms showing the target region of sgPtenQ245* (PCR products were subcloned for clarity). Arrowheads highlight cytosines of the protospacer that show base editing. In the lower example the gene was inactivated by indels at both alleles, while in the upper one by Q245* base editing in one allele and a deletion at the second copy of the gene.


Figure 4Sanger - sequencing chromatograms showing the target region of sgTrp53Q97 * in wild - type ( WT ) and base edited ( BE ) cells . Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3 - expressing NIH3T3 cells with Lenti - sgTrp53Q97 EditR was used to calculate the frequency ( % ) of C - to - T conversion at C8 of the protospacer targeted by sgTrp53Q97 * in BE3 - expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors . Kaplan - Meier curves showing mammary tumor - specific survival for the different models . WapCre ; Brca1F / F ; Trp53F / + ; Col1a1invCAG - BE3 / + females injected with Lenti - sgPik3caE545K / sgTrp53Q97 * - Myc ( n = 6 ) showed a reduced mammary tumor - specific survival compared to animals injected with Lenti - sgTrp53Q97 * - Myc ( n = 5 ) vectors ( 76 days after injection vs 101 days after injection , P < 0 . 05 by Mantel - Cox test ) . Females injected with Lenti - sgNT - Myc ( n = 11 ) did not develop any palpable tumors during the 150 days observation period . BE Analyzer was used to assess from next - generation sequencing data the fraction of wild - type Trp53 alleles , base edited alleles or alleles with indels in tumors from WapCre ; Brca1F / F ; Trp53F / + ; Col1a1invCAG - BE3 / + animals injected with Lenti - sgTrp53Q97 * - Myc . Tumors from WB1P - BE3 animals injected with Lenti - sgNT - Myc mice were used as control . TIDE analysis showing the spectrum of indels of the targeted Trp53 alleles in two independent representative tumors from WapCre ; Brca1F / F ; Trp53F / + ; Col1a1invCAG - BE3 / + mice injected with Lenti - sgTrp53Q97 * - Myc . For the two tumors shown in ( F ) , Sanger - sequencing chromatograms showing the target region of sgTrp53Q97 * . Arrowheads highlight cytosines of the protospacer that show base editing . In the lower example the gene was inactivated by a deletion , while in the upper one by Q97 * base editing . Of note , the allele with the indel also displays base editing at C6 of the protospacer .

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

Figure 4Sanger-sequencing chromatograms showing the target region of sgTrp53Q97* in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgTrp53Q97 EditR was used to calculate the frequency (%) of C-to-T conversion at C8 of the protospacer targeted by sgTrp53Q97* in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ females injected with Lenti-sgPik3caE545K/sgTrp53Q97*-Myc (n=6) showed a reduced mammary tumor-specific survival compared to animals injected with Lenti-sgTrp53Q97*-Myc (n=5) vectors (76 days after injection vs 101 days after injection, P < 0.05 by Mantel-Cox test). Females injected with Lenti-sgNT-Myc (n=11) did not develop any palpable tumors during the 150 days observation period.BE Analyzer was used to assess from next-generation sequencing data the fraction of wild-type Trp53 alleles, base edited alleles or alleles with indels in tumors from WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ animals injected with Lenti-sgTrp53Q97*-Myc. Tumors from WB1P-BE3 animals injected with Lenti-sgNT-Myc mice were used as control. TIDE analysis showing the spectrum of indels of the targeted Trp53 alleles in two independent representative tumors from WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ mice injected with Lenti-sgTrp53Q97*-Myc. For the two tumors shown in (F), Sanger-sequencing chromatograms showing the target region of sgTrp53Q97*. Arrowheads highlight cytosines of the protospacer that show base editing. In the lower example the gene was inactivated by a deletion, while in the upper one by Q97* base editing. Of note, the allele with the indel also displays base editing at C6 of the protospacer.


Figure 1 ( A ) Western blot of SMC3 - LAP expressing HeLa cells treated with siRNA as indicated and synchronized in G2 - phase . Efficiency of RNA interference and SMC3 acetylation levels were analyzed from total extracts and after chromatin fractionation ( asterisks indicate unspecific signals that were depleted from antibody dilutions used in later experiments , presumably by repeated usage of the antibody samples ) . Note that ESCO1 levels were increased on chromatin after depletion of ESCO2 , raising the possibility that a decrease in chromatin bound ESCO2 might be compensated for by ESCO1 recruitment to chromatin . ( B ) Still images of an inverse fluorescence recovery after photobleaching ( iFRAP ) experiment . Fluorescence intensities are false - colored as indicated . Size bar , 5 μm . ( C ) Graph depicting the normalized intensity after photobleaching from two biological replicates to quantify chromatin dissociation kinetics . Error bars denote standard error of the mean ( s . e . m . ) , n = 10 cells per condition . ( D ) Quantification of the relative abundance of SMC3 - LAP in the stable chromatin binding mode .

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

Figure 1(A) Western blot of SMC3-LAP expressing HeLa cells treated with siRNA as indicated and synchronized in G2-phase. Efficiency of RNA interference and SMC3 acetylation levels were analyzed from total extracts and after chromatin fractionation (asterisks indicate unspecific signals that were depleted from antibody dilutions used in later experiments, presumably by repeated usage of the antibody samples). Note that ESCO1 levels were increased on chromatin after depletion of ESCO2, raising the possibility that a decrease in chromatin bound ESCO2 might be compensated for by ESCO1 recruitment to chromatin.(B) Still images of an inverse fluorescence recovery after photobleaching (iFRAP) experiment. Fluorescence intensities are false-colored as indicated. Size bar, 5 μm.(C) Graph depicting the normalized intensity after photobleaching from two biological replicates to quantify chromatin dissociation kinetics. Error bars denote standard error of the mean (s.e.m.), n = 10 cells per condition.(D) Quantification of the relative abundance of SMC3-LAP in the stable chromatin binding mode.


Figure 2 ( A ) Quantification of iFRAP experiments using HeLa cells expressing SMC3 - LAP in the wild - type form ( wt ) or with lysines 105 - 106 mutated to glutamines ( QQ ) or arginines ( RR ) and synchronized in G1 - phase . Error bars denote s . e . m . , n > 25 cells per condition . ( B ) Quantification of chromatin residence time of SMC3 - LAP alleles in G1 - phase . ( C ) Quantification of iFRAP experiments using cells synchronized in G2 - phase . Error bars denote s . e . m . , n > 26 cells per condition . ( D ) Quantification of stably bound SMC3 - LAP alleles in G2 - phase . ( E ) Western blot of mouse fibroblasts expressing SMC3 - LAP in the presence or absence of endogenous Smc3 after gene deletion . Extracts were prepared after subculturing two times . Cells without SMC3 - LAP do not proliferate after deletion of Smc3 . ( F ) Western blot showing HeLa cell extracts after treatment with siRNA as indicated and synchronized in G2 - phase . Note that ESCO1 immunoblot signals were also reduced in sororin depleted cells . We currently do not know if this is an effect of sororin depletion , an off - target effect or an artefact of unequal sample loading . ( G ) iFRAP quantification of stably chromatin bound SMC3 - LAP after siRNA treatment using cells synchronized in G2 - phase . Error bars denote s . e . m . , n > 11 cells per condition .

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

Figure 2(A) Quantification of iFRAP experiments using HeLa cells expressing SMC3-LAP in the wild-type form (wt) or with lysines 105-106 mutated to glutamines (QQ) or arginines (RR) and synchronized in G1-phase. Error bars denote s.e.m., n > 25 cells per condition.(B) Quantification of chromatin residence time of SMC3-LAP alleles in G1-phase.(C) Quantification of iFRAP experiments using cells synchronized in G2-phase. Error bars denote s.e.m., n > 26 cells per condition.(D) Quantification of stably bound SMC3-LAP alleles in G2-phase.(E) Western blot of mouse fibroblasts expressing SMC3-LAP in the presence or absence of endogenous Smc3 after gene deletion. Extracts were prepared after subculturing two times. Cells without SMC3-LAP do not proliferate after deletion of Smc3.(F) Western blot showing HeLa cell extracts after treatment with siRNA as indicated and synchronized in G2-phase. Note that ESCO1 immunoblot signals were also reduced in sororin depleted cells. We currently do not know if this is an effect of sororin depletion, an off-target effect or an artefact of unequal sample loading.(G) iFRAP quantification of stably chromatin bound SMC3-LAP after siRNA treatment using cells synchronized in G2-phase. Error bars denote s.e.m., n > 11 cells per condition.